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Gilmore MS, Clewell DB, Ike Y, et al., editors. Enterococci: From Commensals to Leading Causes of Drug Resistant Infection [Internet]. Boston: Massachusetts Eye and Ear Infirmary; 2014-.

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Enterococci: From Commensals to Leading Causes of Drug Resistant Infection [Internet].

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Pathogenesis and Models of Enterococcal Infection

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Introduction

The vast majority of enterococci, including species that are major agents of nosocomial infection, are peaceful inhabitants of the gastrointestinal (GI) tracts of animals that range from insects to humans. However, situations arise in which enterococci can cause serious disease. The era of modern medicine has created circumstances that facilitate the pathogenic behavior of these microbes in the following ways. First, the use, and arguable overuse, of antibiotics has selected for bacteria that are resistant to these medicines. Even though enterococci may not be as inherently virulent as some other bacterial pathogens, they are facile at collecting and exchanging antibiotic resistance determinants, in addition to being naturally resistant to many antibitoics. Such properties give enterococci a selective advantage in environments with heavy antibiotic usage, such as the hospital, and may allow them to out-compete other species that would normally keep them in check. Another factor is the increasing susceptible population of immunocompromised individuals, which includes the elderly, solid organ and bone marrow transplant patients, and cancer patients. The action of the immune system likely contributes to the commensal balance, and a severely weakened defense system may be unable to keep these species in check. Finally, there are genetic factors that contribute to the ability of enterococci to survive and cause infection in a host environment, which are defined as virulence determinants for the purpose of this discussion. Some of these factors are part of the core genome, while others are traits that can be acquired and shared. Much work has been done over the last 20 years to identify these virulence determinants, and to characterize their mechanisms of action, which is is the main focus of this chapter.

Animal models have made critical contributions to our understanding of the pathogenesis of enterococcal infection. A number of advances recently have been made that allow for new and more facile models to be employed in these studies. However, since the motivation behind a given study usually relates to some aspect of human infection, it is important to understand the parallels and limitations of each model, in order to know the extent to which the results can be extrapolated to humans.

Models for Studying Enterococcal Infection

Models of infection have been used to test the infectivity of enterococci since 1899, when MacCallum and Hastings (MacCallum & Hastings, 1899) infected mice and rabbits with enterococci in an attempt to test Koch’s postulates. Both mice and rabbits succumbed when injected intravenously and intraperitoneally with large inocula of human-derived enterococci (MacCallum & Hastings, 1899). However, their study also illustrated some of the challenges inherent in all investigations of bacterial pathogenesis, i.e., the varying infectivity of different strains, and the varying susceptibility of different hosts. Since this first study, mice and rabbits have served as the most common infection models for studies of enterococcal pathogenesis.

In the last decade, invertebrate models of enterococcal infection have been developed as a complement to mammalian models. The use of invertebrates, such as the nematode Caenorhabditis elegans and the Greater Wax Moth caterpillar Galleria mellonella, has provided new options for pathogenesis studies, in that experimenting with these organisms can be inexpensive and practical. In addition, the large-scale early-phase screens needed for the identification of virulence factors or new therapeutics is prohibitively difficult in mammalian models due to the large numbers of animals required, but can easily be carried out in these small invertebrates (Garsin, et al., 2001; Maadani, Fox, Mylonakis, & Garsin, 2007). In the invertebrates that are amenable to genetic manipulation, it is also possible to examine the host response, as has been done by transcriptional profiling of C. elegans infected with E. faecalis (Englemann, et al., 2011; Wong, Bazopoulou, Pujol, Tavernarakis, & Ewbank, 2007). However, a crucial requirement for interpreting the results from any animal model is a good understanding of the model’s limitations.

Invertebrates

C. elegans

C. elegans is a well-characterized model organism that has been used to elucidate important biological processes in development, gerontology, and, more recently, host-pathogen interactions. It is a small, soil-dwelling nematode that ingests microbes found on rotting fruit as a food source. Presumably because this lifestyle exposes the animal to a variety of potentially pathogenic organisms, C. elegans has evolved an intricate innate immune system that shares many features with higher animals (Ewbank & Zugasti, 2011; Irazoqui, Urbach, & Ausubel, 2010; Tan & Shapira, 2011). Structural barriers, such as a tough collagenous cuticle, protect the epidermal surface from direct penetration by all but a few highly specialized pathogens. A variety of antimicrobial peptides including lysozymes, caenopores, lectins, and ABFs (antibacterial factors) are secreted by the intestinal epithelial cells, while NLPs (neuropeptide-like peptide) and caenacins are secreted by the epidermis (Ewbank & Zugasti, 2011). The intestine (and possibly the epidermis) also releases reactive oxygen species (ROS) as part of the defense response (Chávez, Mohri-Shiomi, & Garsin, 2009; Chávez, Mohri-Shiomo, Maadani, Vega, & Garsin, 2007). Several highly conserved metazoan signaling pathways orchestrate the C. elegans innate immune response including components of the insulin signaling, p38 MAPK, and β-catenin signaling pathways (Irazoqui, Urbach, & Ausubel, 2010). When comparing the innate immune response of C. elegans to the mammalian immune response, it is important to keep in mind that C. elegans does not have an adaptive immune system, nor does it have a cellular aspect to its innate immune response. These differences limit the types of comparisons that can be made between microbial interactions with C. elegans, on one hand, and microbial interactions with mammals, on the other (Ewbank & Zugasti, 2011; Irazoqui, Urbach, & Ausubel, 2010; Tan & Shapira, 2011).

In addition to the fact that C. elegans can be infected by a large array of human pathogens (Powell & Ausubel, 2008), the animal has many characteristics that facilitate the study of such infections. C. elegans are easy to maintain in the laboratory due to their small size (the adult is 1 mM long) and short generation time of three days. They possess a hermaphroditic reproductive style that allows one adult animal to produce up to 300 genetically identical progeny, but also a male sex, generated at a low frequency, that allows for the crossing of strains. The animal has a fully sequenced genome, is easily subjected to RNAi-mediated knockdown of genes, and many mutants and genetic tools exist (Hope, 1999). Because of these features, large-scale screens of both host and pathogen factors that contribute to infection, as well as screens for novel antimicrobials, have been possible (Kurz & Ewbank, 2007).

Host-pathogen interactions with various human infectious agents, including E. faecalis, can be studied easily in C. elegans by exploiting its propensity to feed on a wide range of microbes including bacteria and yeasts (Powell & Ausubel, 2008). The consumption of E. faecalis results in an infection in the lumen of the animal’s intestine that eventually causes its demise. To inoculate C. elegans with E. faecalis by feeding, L4 stage larvae are transferred from a lawn of their normal laboratory food, E. coli strain OP50, to plates of solid agar medium that contain a lawn of E. faecalis (Garsin, et al., 2001). Brain heart infusion (BHI) is the medium most often used, but for certain expression profiling studies in C. elegans, E. faecalis has also been placed on nematode growth medium (NGM), which is a minimal medium that causes slower killing kinetics (Englemann, et al., 2011; Wong, Bazopoulou, Pujol, Tavernarakis, & Ewbank, 2007). Adding appropriate antibiotics prevents contamination by OP50, which is carried over when C. elegans are transferred to E. faecalis. After transfer, the animals die with an LT50 of approximately 4–5 days, depending on the particular E. faecalis strain, medium, and temperature (Garsin, et al., 2001). Usually C. elegans are examined daily and any dead animals are removed from the plates. The animals can be followed on the agar plates, or alternately, they can be moved into liquid culture after an initial inoculation period of 8–12 hours. A liquid medium that was empirically determined to be efficacious for E. faecalis-mediated killing is 10–20% BHI diluted in M9 medium (Moy, et al., 2006). Liquid medium is most commonly used in studies that examine the effects of different drugs on the infectious process — for example, screens for new antimicrobials (Moy, et al., 2006; Moy, et al., 2009).

Experiments that examined the features of an E. faecalis infection of C. elegans showed that there is both colonization and growth inside the intestinal lumen, such that each C. elegans eventually contains 104 to 105 CFU. Invasion of the intestinal cells or the rest of the animal has not been reported. The infection is persistent in that transferring E. faecalis-exposed C. elegans to a lawn of non-pathogenic bacteria does not displace the E. faecalis, and the animals continue to die (Garsin, et al., 2001; Moy, et al., 2006). Infecting C. elegans with a small inoculum of E. faecalis (by mixing it with a bacterium that does not kill C. elegans, such as particular strains of Enterococcus faecium, as described below) still results in the eventual engorgement of the intestine and the demise of the animals. Infection by E. faecalis requires the ability to divide, and E. faecalis exposed to bacteriostatic antibiotics will not kill C. elegans (Garsin, et al., 2001). Electron microscopy allows for the observation of actively dividing E. faecalis cells in the C. elegans intestine (Figure 1). Importantly, C. elegans does not reproduce on E. faecalis. The animals lay eggs, but few hatch, and these few hatchlings almost never grow beyond the initial L1 larval stage (Garsin, et al., 2001). It is not known whether C. elegans larvae are unable to extract nutrients from E. faecalis or whether E. faecalis kills them.

Figure 1. . Electron micrographs of C.

Figure 1.

Electron micrographs of C. elegans intestine from animals infected with E. faecalis for 48 hours (B), compared to uninfected animals (A). Note that the infected animals display a distended intestinal lumen, erosion of the microvilli, and colonization (more...)

In contrast to E. faecalis, many strains of E. faecium grown aerobically colonize, but do not grow or persist within the C. elegans gut, and do not kill the animal (Garsin, et al., 2001). However, when E. faecium is cultured anaerobically and then restored to aerobic conditions, E. faecium kills the nematodes with an LT50 of 4–6 hours, due to hydrogen peroxide production by the bacterium (Moy, Mylonakis, Calderwood, & Ausubel, 2004). The mechanism by which E. faecium produces H2O2 is related to its exclusive use of glycolysis and fermentation, which unlike E. faecalis metabolism, can involve a partial electron transport chain (see The physiology and metabolism of enterococci). These metabolic features under aerobic conditions cause E. faecium to regenerate NAD+ by water- and peroxide-forming NADH oxidases (Huycke M. M., 2002). Moy et al. (Moy, Mylonakis, Calderwood, & Ausubel, 2004) provided evidence that these oxidases generate the H2O2 observed. Normally, an NADH peroxidase degrades the H2O2, which prevents its accumulation. Moy et al. speculated that the gene for this enzyme is expressed only under aerobic conditions, such that NADH peroxidase levels are low when transitioning the plates from anaerobic to aerobic conditions, which allows for significant amounts of H2O2 to accumulate (Moy, Mylonakis, Calderwood, & Ausubel, 2004).

The C. elegans model has been used to study and discover factors with a possible role in E. faecalis virulence. Cytolysin and the Fsr system affect virulence in a wide variety of hosts, and C. elegans is no exception. The presence of cytolysin significantly increases the rate of killing, both on solid and in liquid medium (Garsin, et al., 2001; Moy, et al., 2006). The Fsr system and two of the proteases it regulates, gelatinase and serine protease, also affect virulence in the C. elegans model (Garsin, et al., 2001; Moy, et al., 2006; Sifri, et al., 2002). Response to nutritional stress regulated by the stringent response is another important virulence-related factor for optimal E. faecalis infection in C. elegans, as the loss of both ppGpp synthases that control the stringent response, RelA and RelQ, limits colonization and infection (Abranches, et al., 2009). Somewhat surprisingly, many surface proteins with possible functions in attachment or adhesion, such as Esp, Ace, Acm, and EbpA, do not play essential roles in the C. elegans infection model, perhaps as a consequence of functional redundancy. Mutations in the corresponding genes have not been identified in genetic screens and no attenuated phenotypes have been observed in direct testing ((Maadani, Fox, Mylonakis, & Garsin, 2007), Garsin lab, unpublished data). However, the loss of ef3314, which encodes a surface protein with some structural similarity to Esp, does significantly attenuate virulence and suggests that some surface factors do play essential roles in C. elegans (Creti, et al., 2009). A forward genetic screen of E. faecalis transposon mutants for defects in killing C. elegans uncovered potential virulence determinants with predicted functions in transcription, metabolism, and damage control. Five of nine mutants tested were attenuated in a mouse peritonitis model, which demonstrates the utility of using this invertebrate model to identify E. faecalis genes that are involved in mammalian infection (Maadani, Fox, Mylonakis, & Garsin, 2007).

Another exciting use of C. elegans is as a tool to identify low molecular-weight compounds that are protective against E. faecalis infection. To develop this model, it was first demonstrated that traditional antibiotics such as tetracycline and vancomycin at clinically relevant concentrations cure C. elegans of an E. faecalis infection (Moy, et al., 2006). Then various libraries of compounds were tested for the ability to protect C. elegans from E. faecalis by screening for increased survival in a liquid infection model. As a result, two compound screens have been described. A small scale screen of about 7,000 compounds, in which curing was scored manually by visual inspection, provided proof of principal for the approach (Moy, et al., 2006). The second screen of approximately 37,000 compounds used robotic liquid-handling to set up the assay, including distribution of C. elegans into 384 well assay plates, and automated microscopy and image analysis to score the animals for mortality (Moy, et al., 2009). An unexpected and potentially important outcome of these screens is that many of the compounds identified did not prevent in vitro E. faecalis growth at the concentrations that were found to be protective of C. elegans. Many compounds were protective at 10- to 100-fold lower concentrations than their corresponding in vitro MICs for E. faecalis. This is in contrast to traditional antimicrobials, in which the concentration that exerts an inhibitory effect on in vitro bacterial growth is significantly lower than the concentration required at the site of infection to achieve clearance in animal models and human patients.

How these drugs are acting to protect C. elegans from E. faecalis-mediated killing, and whether or not the same effects will be observed in vertebrate models of infection, are areas of active study. Some hypotheses include that these compounds exert their effects on the host innate immune system, block bacterial virulence gene expression, and/or are modified into a more active form within the host (Moy, et al., 2006; Moy, et al., 2009). Recent work has shown that one compound (referred to as RPW-24) activates the C. elegans p38 MAPK pathway, thereby conferring resistance not only to E. faecalis but also to P. aeruginosa infection (Pukkila-Worley, et al., 2012). The ability to use C. elegans infected with E. faecalis as a model to identify compounds that protect against infection could lead to new classes of anti-infectives of clinical importance, and the continuation of this work will be followed with much interest.

Insects

Insects, particularly the larva of the Greater Wax Moth Caterpillar, Galleria mellonella, have recently emerged as important models for testing the roles of potential virulence factors in infectious human pathogens, including enterococci. Insects have surprisingly complex innate immune systems, based on both cellular and humoral mechanisms with parallels in mammals. The insect blood equivalent, called the hemolymph, contains innate immune cells called haemocytes, which can phagocytose invading pathogens in a manner similar to human macrophages and neutrophils. Though insects do not generate specific antibodies, they do produce a wide range of substances that constitute the humoral response. The factors include components of the clotting and melanization cascades and various anti-microbial peptides, some of which are shared with vertebrates (lysozymes, metalloproteinases, defensins) (Kavanagh & Reeves, 2004).

G. mellonella is an attractive model, because larvae can easily be reared at low cost or cheaply obtained from commercial sources. The animals are infected by injection into the hemolymph and survival is monitored over a few days, with death usually measured as the endpoint. Staff can also be quickly and easily trained in the procedures. Limitations to the model include the lack of a sequenced genome, the inability to genetically manipulate the organism because no molecular tools are currently available, and sometimes, an imperfect recapitulation of pathogen-human interactions (Mylonakis E. , 2008; Olsen, Watkins, Cantu, Beres, & Musser, 2011).

G. mellonella has only recently been used as a model for E. faecalis infection. E. faecalis, E. faecium, and other species of enterococci are naturally found to be associated with insects (Cox & Gilmore, 2007; Martin & Mundt, 1972). The use of G. mellonella as an infection model began when a group isolated a strain of E. faecalis from larvae that died of a bacterial infection. Injection of this strain back into hemolymph of the animal resulted in death (Park, Kim, Lee, Seo, & Lee, 2007). In other work, a dose-dependent sensitivity to infection by E. faecalis was discovered (Yan, et al., 2009). Depending on the strain of E. faecalis being used, investigators observed death within one to two days of inoculating with anywhere between 1 x 105 to 5 x 108 CFUs (Michaux, et al., 2011; Yan, et al., 2009; Zhao, et al., 2010). E. faecium and E. durans are significantly less infective in this model (Gaspar, et al., 2009). Only large inocula (1 x 108) of some strains of E. faecium resulted in caterpillar mortality. However, at lower inoculums (1 x 106), better colonization was observed among some hospital-adapted isolates of E. faecium (Lebreton, et al., 2011). Infection by oral feeding of E. faecalis was shown to be ineffective, which is perhaps not surprising when considering E. faecalis’s common role as a gut commensal (Fedhila, et al., 2010).

Several virulence factors affect time to death in G. mellonella larvae when mutants that carry lesions in the genes-of-interest are compared to their isogenic, wild type controls. Not surprisingly, the Fsr quorum-sensing system and specifically one of the genes it regulates, gelE, were discovered to be important. Park et al. (Park, Kim, Lee, Seo, & Lee, 2007) observed that a secreted substance had insecticidal activity, and purification identified it as GelE. They then demonstrated that GelE could digest cecropin and other antimicrobial peptides produced by G. mellonella (Park, Kim, Lee, Seo, & Lee, 2007). Gaspar et al. (Gaspar, et al., 2009) showed that strains that lacked gelE were less virulent, as compared to isogenic controls. The Fsr quorum-sensing system is generally necessary for gelE expression, and loss of fsrB caused attenuated phenotypes in two E. faecalis strains, but not in the OG1RF background—perhaps because it has residual gelatinase activity (Gaspar, et al., 2009; Singh K. V., Nallapareddy, Nannini, & Murray, 2005). Loss of the ppGpp synthase, relA, which is critical for stress adaption, was shown to attenuate virulence in G. mellonella. A more stress-resistant mutant that consisted of a C-terminal truncation was more virulent (Yan, et al., 2009). Loss of another stress resistance factor, the heat-shock protein ClpB, also attenuated virulence (de Olivera, et al., 2011). The collagen- and laminin-binding protein Ace resulted in attenuated infection in G. mellonella when the gene was deleted (Lebreton, et al., 2009). Other E. faecalis factors that affect virulence in this model include methionine sulfoxide reductases (encoded by msrA and msrB) and the transcription factor SlyA, whose loss actually increases virulence. The methionine sulfoxide reductases and SlyA have also been shown to affect persistence within mouse organs following IV injection and survival within peritoneal macrophages, which demonstrates that their importance during infection extends beyond G. mellonella (Michaux, et al., 2011; Zhao, et al., 2010).

In addition to examining specific potential virulence determinants, G. mellonella is being used to identify new ones. One group looked at the ways in which lyosogeny with different phages affects virulence. The authors observed changes in G. mellonella survival, both increased and decreased, that were dependent upon the phage employed. Though the genetic determinants have not yet been identified, these experiments suggest that the phages are introducing or disrupting factors that affect virulence (Yasmin, et al., 2010). Additionally, G. mellonella was employed in a screen for in vivo activated genes using recombination-based in vivo expression technology (R-IVET). Among the genes found, the inactivation of a two-component system, ef_3196 and ef_3197, resulted in a virulence defect, whereas deletion of a gene encoding an ankyrin repeat protein caused a hypervirulent phenotype (Hanin, et al., 2010). The G. mellonella model has also been demonstrated to be a powerful tool for large-scale investigations, such as the screening of a targeted E. faecalis mutant library for new fitness factors (Rigottier-Gois, et al., 2011).

E. faecalis was first reported to infect Drosophila melanogaster in 2001. Infection of the fly was achieved by pricking the animal with a needle previously dipped into a concentrated culture of bacteria. The goal of this investigation, and most of the Drosophila studies afterward, was to elucidate host immune responses to a Gram-positive agent, rather than to understand E. faecalis virulence mechanisms (Michel, Reichhart, Hoffmann, & Royet, 2001).

Cox and colleagues (Cox & Gilmore, 2007) characterized the gut microbiota of wild-caught and laboratory reared Drosophila, and localized enterococci within that consortium. Enterococci were among the most abundant gut microbes found using sequence-based metagenomics. Feeding Drosophila strains that were cytolytic resulted in increased mortality (40% mortality vs. 12%), which demonstrated that this virulence factor could compromise this host by a natural infection route (Cox & Gilmore, 2007).

Vertebrates

In contrast to infection studies carried out in invertebrates, vertebrate animal infection models afford researchers the ability to study enterococcal pathogenesis in the context of the innate and adaptive immune responses, and to evaluate bacterial effects on organs that closely resemble those found in humans. An important challenge in attempting to approximate human infection in a model is that enterococcal strains of interest are either commensals or are recently derived from commensals, and they infrequently infect humans or animals with intact immune systems. Therefore, to achieve a progressive infection using healthy vertebrate animals, large inocula, either with or without foreign bodies (such as intravascular catheters for the endocarditis model) are needed. Some models have been developed that induce neutropenia in animals prior to infection (Griffith, Rodriguez, Corcoran, & Dudley, 2008; Leendertse M. , et al., 2009; Onyeji, Nicolau, Nightingale, & Bow, 2000) or that take advantage of naturally immune limited tissues (such as the endophthalmitis model (Callegan, Booth, Jett, & Gilmore, 1999; Jett, Atkuri, & Gilmore, 1998; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992) further discussed below), which enables the production of disease manifestations using fewer organisms. Although these models are generally more technically demanding, the use of animal models of enterococcal infection has greatly enhanced our understanding of the chromosomal- and plasmid-encoded determinants that enable enterococci to cause disease, as well as the pathogenesis of those infections in mammalian tissues.

Endocarditis

Enterococci are the third most common cause of endocarditis (Murdoch, et al., 2009), which is one of the most life-threatening infections caused by E. faecalis and E. faecium, and as a result, using a mammailian model to elucidate the role of enterococcal virulence traits is critical to derive new approaches for treatment and prevention. Enterococcal endocarditis involves the formation of a biofilm on heart valves at sites of damage that become integrated into masses called vegetations. A number of enterococcal adhesins or proteins known to function in biofilm formation have been tested in endocarditis models. Among those that contribute to E. faecalis endocarditis virulence are gelatinase (Thurlow L. R., et al., 2010), the protease Eep (Frank, et al., 2012), aggregation substance (Chow, et al., 1993; Chuang, et al., 2009), Ebp pili (Nallapareddy S. R., et al., 2006), and Ace (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010). Endocarditis was observed to be lethal in a rabbit model in the presence of aggregation substance-expressing cells that also expressed the toxin cytolysin, and vegetations were larger when caused by an isogenic strain that expressed aggregation substance alone (Chow, et al., 1993). The severity of endocarditis was reduced in rats that were injected with a recombinant Ace protein or passively immunized with anti-Ace antibodies (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010). Besides the cytolysin toxin, adhesins, and biofilm determinants, the general stress response protein Gls24 was also shown to contribute to E. faecalis endocarditis in rats (Nannini, Teng, Singh, & Murray, 2005). In E. faecium, the adhesin Esp was found to be necessary for full virulence in the rat endocarditis model (Heikens, et al., 2011).

Enterococcal endocarditis models have generally used either Sprague-Dawley rats or New Zealand white rabbits, with a number of procedural variations. In both models, damage to the aortic valve is induced by advancing a catheter into the right carotid artery to the point where it crosses the valve. Catheters are then either left in place for the duration of the infection (Thurlow L. R., et al., 2010), or removed after two hours (Chuang, et al., 2009; Schlievert, et al., 1998). A small study aimed at examining the effect of leaving the catheter in place during infection found that the number and size of vegetations formed by strains was increased, as compared to rabbits from which the catheters had been removed (Schlievert, et al., 1998). This observation led the study authors to propose that vegetation formation in the presence of a transaortic catheter may be artificially enhanced (Schlievert, et al., 1998).

In the rat model, inocula of 107-108 CFU are often administered through the catheter twenty minutes after its placement, the catheter is then heat-sealed, and the end into which microbes were added is embedded in subcutaneous tissue. The animals are typically euthanized at 24 hours post-inoculation (Nallapareddy S. R., et al., 2006). Bacteria may also be injected through the rat tail vein. In rabbits with the catheter remaining throughout infection, bacteria are injected through an ear vein 24 hours after surgery, and the experiment is terminated at 48 hours post-inoculation (Thurlow L. R., et al., 2010). Alternatively, bacteria are injected through an ear vein within two hours following catheter removal, and rabbits are euthanized at 96 hours post-inoculation (Chuang, et al., 2009; Frank, et al., 2012). The reported inoculum used for rabbit experiments was ~107 CFU for strain E. faecalis V583 in animals with retained catheters, and ~109 of OG1SSp or OG1RF in animals with the catheters removed (Chuang, et al., 2009; Frank, et al., 2012; Thurlow L. R., et al., 2010). Following euthanasia, hearts are dissected to expose the aortic valve in order to harvest vegetations for bacterial enumeration by quantitative culture. Bacterial loads in the blood, kidneys, spleen, and liver can also be determined (Nallapareddy S. R., et al., 2006; Thurlow L. R., et al., 2010).

The number of variables that differ between endocarditis models requires that caution be exercised in interpreting the results of these experiments. The differences that should be considered include vegetation formation in the presence or absence of a foreign body, whether bacteria encounter the damaged heart valve by circulation or by direct delivery to the infection site, and how long after valve damage bacteremia is induced. The latter is important because sterile vegetations, or nonbacterial thrombotic lesions, can form on damaged valves over time, such that the delayed onset of enterococcal bacteremia (such as in (Thurlow L. R., et al., 2010)) will result in bacterial colonization of an extant mass on the damaged valve. By comparison, the induction of bacteremia shortly after valve damage likely results in the bacteria being present at the valve concurrent with the onset of vegetation formation. Therefore, it is possible that different genetic determinants may be involved in enterococcal colonization of damaged heart valves, and that discrepancies may arise as to whether a gene contributes to endocarditis virulence, based on the model in which it was tested.

Urinary tract infection

Enterococcal species are the second most common cause of catheter-associated urinary tract infection (CAUTI) reported to the National Healthcare Safety Network over a 22-month period beginning in January 2006 (Hidron, et al., 2008). Several murine models of E. faecalis urinary tract infection (UTI) have been used to study the pathogenesis of infection over the past two decades. Most are variations of ascending unobstructed urinary tract infections and are adapted from models used to study Gram-negative uropathogens (Johnson, Clabots, Hirt, Waters, & Dunny, 2004; Kau, et al., 2005; Shankar, et al., 2001). Typically, 106-108 organisms are administered to anesthetized mice via a transurethral catheter, and animals are sacrificed at intervals ranging from one to fourteen days, at which time urine, the bladder, and the kidneys are harvested for quantitative bacterial culture and histopathology (Kau, et al., 2005; Shankar, et al., 2001; Singh, Nallapareddy, & Murray, 2007). E. faecalis shows a tropism for the kidneys in this type of model, making it useful for studies focused on factors that contribute to enterococcal pyelonephritis (Johnson, Clabots, Hirt, Waters, & Dunny, 2004; Kau, et al., 2005; Shankar, et al., 2001; Singh, Nallapareddy, & Murray, 2007). Infection of C57BL/6J mice with an E. faecalis urinary tract isolate caused an inflammatory response in the kidneys, but not the bladder (Kau, et al., 2005). The inflammatory response was different than that induced by uropathogenic E. coli and was found to be independent of receptor TLR2 (Kau, et al., 2005). Deletion or disruption of the coding sequences for a number of E. faecalis adhesins, including Ace, Esp, Epa, and Ebp pili, resulted in reduced virulence in mouse models of ascending UTI infection (Lebreton, et al., 2009; Shankar, et al., 2001; Singh, Lewis, & Murray, 2009; Singh, Nallapareddy, & Murray, 2007). The E. faecium pilus also contributes to UTI virulence (Sillanpää J. , et al., 2010).

A model that more closely parallels E. faecalis CAUTI has been developed (Guiton, Hung, Hancock, Caparon, & Hultgren, 2010), which involves implantation of silicone catheter segments into the bladders of C57BL/6Ncr mice immediately before inoculation with 107 organisms. Biofilms formed in vivo by strain OG1RF on implanted catheter segments contained 107 CFU/implant, from two to seven days post-infection. The presence of the implants increased bladder colonization by three orders of magnitude at 24 hours post-infection, and increased kidney colonization by more than one order of magnitude between 24 hours and 7 days post-inoculation, as compared to control infections. Implantation of silicone pieces resulted in marked histological damage to the uroepithelium, and caused increased expression of several cytokines, including IL-6, G-CSF, and keratinocyte-derived cytokines. Genetic studies indicate that a sortase A-dependent substrate is necessary for virulence in this model (Guiton, Hung, Hancock, Caparon, & Hultgren, 2010).

Endophthalmitis

Because of the challenges presented in handling immune-compromised animals, and because of the difficulty studying the progression of infection in a closed tissue, such as the heart or peritoneal cavity, an endophthalmitis model was developed (Callegan, Booth, Jett, & Gilmore, 1999; Jett, Atkuri, & Gilmore, 1998; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992) that capitalizes on 1) the immune privileged nature of the interior of the eye and its natural susceptibility to infection; 2) the fact that the infection remains largely localized, and the presence of an uninfected control in the same animal in the contralateral eye; 3) the ability to visually follow the infection using inexpensive and readily available instrumentation (an ophthalmoscope); and 4) the ability to objectively and sensitively measure changes in organ function by using electroretinography. In the rabbit model, which is less technically demanding than a similar mouse model, as few as 1000 enterococci are injected typically (although as few as 10 organisms can seed an infection (Stevens, Jensen, Jett, & Gilmore, 1992)) in a 10 μl volume of PBS through an avascular point in the sclera, called the pars plana, to limit intraocular bleeding. The placement of the inoculum into the vitreous immediately behind the lens allows the process to be followed using a surgical microscope. Because of the gel-like nature of the vitreous, the inoculum forms a visible bubble of PBS that refracts light slightly differently than does vitreous. After about 6 hours, fibrin contained within the vitreous begins to coalesce around the 10 μl bubble, and this can be seen with an ophthalmoscope. The clearest direct view of the microvasculature of the body can be found in the retina. Examination of the retina with an ophthalmoscope after about 12 hours post-infection reveals capillaries in the infected eye that begin to dilate because of the microbe-induced inflammation. As early as 16 hours post-infection, a whitening of the microvessels in the retina can be observed using an ophthalmoscope, as neutrophils begin adhering to the vessel walls. Shortly after that, streams of neutrophils that emanate from the optic nerve head can be seen (Callegan, Booth, Jett, & Gilmore, 1999; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992).

In addition to direct observation, measurements routinely performed during the course of infection include assessment of retinal function by electroretinography (ERG). This involves specialized equipment for assessing electrophysiological behavior of neural cells of the retina. In brief, a fine gold electrode is placed on the surface of the eye and a grounding electrode is placed elsewhere on the anaesthetized animal. It is then exposed to a series of xenon strobe flashes. As the photoreceptors and interconnecting neurons fire, an electrical potential is generated, which is detected by the electrodes. The magnitude of the potential is proportional to the number of cells firing. Using this tool, a decline in the function of the photoreceptor layer can be measured, from 100% functionality to 0%, typically over the course of a 48–72 hour experiment. Following termination of the study and dissection of the eye, other parameters can be measured, including enumeration of inflammatory cells in the cornea, anterior chamber, vitreous and retina, as well as histopathological examination of changes in the vitreous and the architecture of the retina and surrounding structures (Callegan, Booth, Jett, & Gilmore, 1999; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992). Inoculation with approximately 102 organisms leads to a vitreous bacterial load of 108–109 organisms within 24 hours (Callegan, Booth, Jett, & Gilmore, 1999; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992). Changes in these parameters have been used to examine the role of the cytolysin of E. faecalis in the pathogenesis of enterococcal infection, as well as its susceptibility to treatment (Callegan, Booth, Jett, & Gilmore, 1999; Choi, Hahn, Osterhout, & O'Brien, 1996; Jett, Jensen, Atkuri, & Gilmore, 1995; Jett, Jensen, Nordquist, & Gilmore, 1992; Stevens, Jensen, Jett, & Gilmore, 1992; Wada, et al., 2008). Additionally, the contributions to the pathogenesis of infection of the E. faecalis quorum-sensing fsr regulatory locus (Mylonakis, et al., 2002), and its regulated proteases gelatinase and serine proteases (Engelbert, Mylonakis, Ausubel, Calderwood, & Gilmore, 2004) have been studied.

Peritonitis and lethality experiments

One of the most widely used infection models is the intraperitoneal infection/peritonitis model. This typically involves mice or rats and consists of intraperitoneal injection of enterococci, followed by daily monitoring for signs of morbidity or death. Parameters measured, often at pre-determined time points, include bacterial counts and assessment of the host response in peritoneal lavage fluid and organs. Without an adjuvant, high concentrations of organisms, ranging from 107–1011 CFU/ml, are required to achieve LD50. This approach was used in initial studies that compared the relative lethality of strains that expressed cytolysin or aggregation substance (Dupont, Montravers, Mohler, & Carbon, 1998; Ike, Hashimoto, & Clewell, 1984). Singh et al. (Singh, Lewis, & Murray, 2009) found that incorporation of sterile rat fecal extracts into the inoculum lowered the LD50 of strain OG1RF, and this approach was used to evaluate the relative virulence of several strains with mutations in putative virulence determinants (Singh, Qin, Weinstock, & Murray, 1998). The cytolysin was observed to enhance toxicity upon intraperitoneal challenge approximately 100-fold (Singh, Qin, Weinstock, & Murray, 1998). Other E. faecalis traits that lower LD50 values, or reduce times to death upon intraperitoneal challenge include gelE (Singh, Qin, Weinstock, & Murray, 1998), the epa locus (Xu, Singh, Murray, & Weinstock, 2000), fsr genes and sprE (Qin X. , Singh, Weinstock, & Murray, 2000), etaRS, which encodes a two component system (Teng, Wang, Singh, Murray, & Weinstock, 2002), gls24 (Teng, Nannini, & Murray, 2005), and the gene that encodes the transcriptional regulator Ers (Riboulet-Bisson, et al., 2008). A mouse peritonitis model was recently used to screen a recombination-based in vivo expression technology library in order to identify genes that are upregulated during growth in infection (Hanin, et al., 2010).

In addition to studying entrococcal virulence, an E. faecium peritonitis model was established to study the way in which the mouse innate immune system responds to bacterial infection (Leendertse M. , et al., 2008). Peritoneal infection with 108 CFU in healthy mice led to gradual clearing of enterococci over 48 hours, which was accompanied by an initial increase in neutrophils (6 hours) and a later upsurge in macrophages (24–48 hours). By using mice deficient for TLR2 and the TLR common adaptor protein MyD88, the authors showed that bacterial clearance and neutrophil influx were diminished during the first several hours post-inoculation. In a follow-up study (Leendertse M. , et al., 2009), the same group demonstrated that bacterial loads were increased for longer periods of time in mice with induced neutropenia, and that this was associated with increased levels of TNF-alpha and IL-6 and decreased recruitment of macrophages to the peritoneal fluid. Opsonization by complement and the presence of peritoneal macrophages appeared to be necessary for optimal clearance of E. faecium by 48 hours post-infection (Leendertse M. , et al., 2010; Leendertse M. , et al., 2009). This series of experiments suggests that many aspects of the innate immune system are required in a host for maximal protection against E. faecium intra-abdominal infection.

Subcutaneously implanted foreign bodies

Models employing subcutaneously implanted foreign bodies, or tissue cages, have served as a means to study both subdermal abscess formation and implanted device biofilm infections caused by E. faecalis (Frank, et al., 2012; Furustrand Tafin, et al., 2011). Hollow, perforated plastic or Teflon devices are subcutaneously placed in the flank of rabbits (Frank, et al., 2012) or guinea pigs (Furustrand Tafin, et al., 2011) under aseptic conditions, and wounds are allowed to heal for 2–6 weeks. During healing, the implanted chambers are encapsulated by host tissues and fill with fluid. Cages are inoculated by injecting organisms through the cage perforations, and infected cage fluid can be withdrawn at any time during the course of infection. In the rabbit model, it is known that bacteria remain localized within the chamber, rather than escaping to the bloodstream to cause bacteremia, as the animals do not exhibit overt signs of illness in experiments carried out over as many as seven days. The rabbit model has been used to characterize E. faecalis gene expression during growth in a mammalian host through transcriptional microarrays and recombinase-based in vivo expression technology (Frank, et al., 2012; Frank, Lemos, Schlievert, & Dunny, 2012). Since the rabbits do not become outwardly sick, the rabbit subdermal abscess model is not particularly useful for assessing the systemic impact of enterococcal infection, but conclusions can be drawn about persistence in vivo in the context of an activated host immune response (Frank, et al., 2012). The guinea pig model has been shown to be useful for testing antibiotic activity against E. faecalis in both the planktonic and biofilm states (Furustrand Tafin, et al., 2011).

Analysis of Virulence of E. faecalis

Because most enterococcal infections have historically been caused by strains of E. faecalis, and because the virulence of E. faecium strains is more subtle and the strains are often more difficult to manipulate, most virulence studies of enterococci have examined E. faecalis and its factors. Both secreted factors, as well as surface localized properties, have been found to contribute to the severity of infection.

Secreted Factors

The cytolysin

The cytolysin contributes to the virulence of E. faecalis in humans and all animal models tested (268a), and is a structurally novel toxin and member of the lantibiotic class of bacteriocins expressed by many strains of E. faecalis (Haas & Gilmore, 1999; Van Tyne, Martin, & Gilmore, 2013). It was first characterized in the 1930s and was studied for the hemolytic activity that it conferred to group D streptococcal isolates (Todd, 1934). Later, it was noted that hemolytic isolates of S. faecalis (now E. faecalis) also inhibited a broad range of Gram-positive organisms (Brock, Peacher, & Pierson, 1963; Stark, 1960). The simultaneous loss of both bactericidal and hemolytic activities by these strains upon UV exposure, with subsequent reversion, suggested a single determinant that encodes both activities (Brock & Davie, 1963).

The bacteriocin activity of enterococcal strains was examined by Sherwood et al. (Sherwood, Russell, Jay, & Bowman, 1949) who found that of 61 hemolytic streptococci, 17 were found to produce an “antibiotic substance” active against other streptococci. Five of the eight hemolytic group D (enterococcal) strains in this group were also found to be capable of bacteriocin production. Later, Brock et al. (Brock, Peacher, & Pierson, 1963) found that over 50% of enterococcal isolates examined produced some type of bacteriocin. These authors defined five different types of bacteriocins, based on activity spectrum and biochemical characteristics, noting that type 1 bacteriocin was produced by all strains of Streptococcus zymogenes (hemolytic E. faecalis), and that this bacteriocin had a particularly wide spectrum of activity against Gram-positive bacteria.

Epidemiological data supports a role for the cytolysin as a toxin in human infection. Infection-derived isolates of E. faecalis, particularly those that cause multiple infections in a hospital ward, showed enrichment for the cytolysin (Huycke & Gilmore, 1995; Huycke, Spiegel, & Gilmore, 1991; Ike, Hashimoto, & Clewell, 1987). Another study found that cytolysin was as prevalent in hospital-derived fecal samples as clinical samples, but was significantly less common in community fecal samples, which suggests that the hospital environment selects for cytolysin (Coque, Patterson, Steckelberg, & Murray, 1995). The cytolysin has been associated with lethality to humans following analysis of an outbreak of multiple antibiotic-resistant E. faecalis (Huycke, Spiegel, & Gilmore, 1991). Patients infected with cytolytic, gentamicin/kanamycin-resistant strains were found to be at a five-fold increased risk of an acutely terminal outcome. Another large prospective study of 398 patients with enterococcal bacteremia, however, did not find a statistically significant correlation between 14-day mortality and gelatinase, cytolysin or Esp, either singly or in combination (Vergis, et al., 2002).

In well controlled animal models, ranging from mammals to invertebrates, the cytolysin makes a readily demonstrable contribution to the severity of infection (Chow, et al., 1993; Garsin, et al., 2001; Huycke, Spiegel, & Gilmore, 1991; Ike, Hashimoto, & Clewell, 1984; Ike, Hashimoto, & Clewell, 1987; Jett, Jensen, Nordquist, & Gilmore, 1992). In most of these studies, the role of the cytolysin was evaluated using isogenic mutants of the cytolysin operon. The first demonstration of the toxigenic activity of the cytolysin was presented by Ike et al. (Ike, Hashimoto, & Clewell, 1984), who employed murine lethality tests to show that E. faecalis strains that express the cytolysin are an order of magnitude more toxic than isogenic, non-cytolytic strains. Also, a hypercytolytic strain derived by a Tn917 insertion in the cytolysin operon resulted in a further two-fold increase in toxicity. Using different strains of enterococci, others have confirmed that the cytolysin contributes to the 50% lethal dose (LD50), increasing lethality by one or more orders of magnitude (Dupont, Montravers, Mohler, & Carbon, 1998; Singh, Qin, Weinstock, & Murray, 1998).

The role of cytolysin in the pathogenesis of E. faecalis infectious endocarditis was assessed in a rabbit model (Chow, et al., 1993). By examining E. faecalis strains that expressed either cytolysin and/or aggregation substance, it was determined that these factors had a synergistic effect on rabbit mortality. Mortality was about 15% in rabbits infected with strains that expressed aggregation substance alone, whereas mortality jumped to 55% when both aggregation substance and cytolysin were expressed. In contrast, the lethal effect of the cytolysin in this model was essentially abrogated in the absence of aggregation substance. In retrospect, these results are not altogether surprising, given that we now know that the expression of cytolysin is quorum-dependent, and aggregation substance most likely facilitates E. faecalis adherence at the infection site, which promotes the manifestation of the toxic effects of the cytolysin (Haas, Shepard, & Gilmore, 2002).

E. faecalis is frequently implicated in surgical site infections, including post-operative endophthalmitis, which is often associated with poor visual outcomes (Callegan, Engelbert, Parke III, Jett, & Gilmore, 2002). In a rabbit model of experimental endophthalmitis, the E. faecalis cytolysin was found to contribute significantly to ocular virulence. Infection with a cytolytic E. faecalis strain, using as few as 100 organisms, was associated with a rapid loss of vision and severe retinal tissue damage, both of which were not observed upon infection with a mutant strain that was defective in cytolysin production (Jett, Jensen, Nordquist, & Gilmore, 1992). Furthermore, combined antimicrobial and anti-inflammatory therapy that allowed for recovery in rabbits infected with a non-cytolytic mutant proved completely ineffective in the treatment of rabbits infected with an isogenic, cytolytic strain (Jett, Jensen, Atkuri, & Gilmore, 1995).

Given that one natural habitat of the enterococci is the gastrointestinal tract, it has been of interest to see which enterococcal factors might promote translocation across the intestinal epithelium. With regard to the cytolysin, although a direct comparison between isogenic strains differing in cytolysin production was not made, Wells et al. (Wells, Jechorek, & Erlandsen, 1990) demonstrated high levels of translocation by a cytolytic E. faecalis strain. Concentrations of E. faecalis in this study ranged from 109 to 1010, which suggests conditions that would enable the production of cytolysin. Huycke et al. (Huycke, Joyce, & Gilmore, 1995) observed that during in vitro growth of a 1:1 mixture of cytolytic and isogenic non-cytolytic strains of E. faecalis, the cytolytic strain outgrew the mutant. However, in a murine colonization model achieved by orogastric administration to antibiotic-treated mice, a 1:1 mixture of cytolytic and non-cytolytic E. faecalis strains resulted in equal colonization, as detected by measuring the CFUs from the stools of mice after one or seven days. As with negative results from all models, however, it is difficult to know in this case whether these results mean that the bacteriocin activity of the cytolysin does not contribute to colonization of hospitalized patients, or whether the test as conducted does not fully model the ecological conditions and competing flora that exist in the gastrointestinal tracts of hospitalized patients.

The contribution of cytolysin to proliferation in the bloodstream has been evaluated. In a peritonitis model evaluating isogenic mutants, about four orders of magnitude greater numbers of cytolytic E. faecalis were quantified in the bloodstream at 48 hours of infection, as compared to the non-cytolytic mutant (Huycke, Sahm, & Gilmore, 1998). Although a mechanism for this observation is not readily apparent, the cytolysin is known to possess activity against macrophages and polymorphonuclear leukocytes (PMNs), which implies a potential role in immune evasion (Miyazaki, et al., 1993). An in vitro study that evaluated PMN-mediated killing of E. faecalis failed to observe differences between cytolytic E. faecalis and isogenic mutants, and it was not clear if cytolysin was indeed expressed under the experimental conditions used for the assay (Arduino, Murray, & Rakita, 1994). Along these lines, the survival of a cytolytic strain of E. faecalis within peritoneal macrophages in vitro was not different from that of a non-cytolytic strain as tested (Gentry-Weeks, Karkhoff-Schweizer, Pikis, Estay, & Keith, 1999).

As mentioned, expression of the cytolysin by E. faecalis resulted in a significant increase in lethality when C. elegans nematodes were fed on a lawn of bacteria (Garsin, et al., 2001). More recently, a study examining the native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis found that high-level colonization with a cytolytic strain of E. faecalis increased killing of Drosophila, as compared to an isogenic non-cytolytic strain (Cox & Gilmore, 2007).

In summary, a wide variety of infection models have shown a role for the cytolysin in E. faecalis virulence. The broad activity of the cytolysin against both prokaryotic and eukaryotic cell types suggests that cytolytic activity might provide access to key nutrients that are not accessible to non-cytolytic strains. Supporting this hypothesis, it has been shown that E. faecalis is capable of assembling an electron transport chain through the synthesis of b-, d- and o-type cytochromes (Pritchard & Wimpenny, 1978; Ritchey & Seeley Jun, 1974). Assembly of these cytochromes requires the uptake of exogenous hemin, which could presumably become available through lysis of erythrocytes or other target cells. The net effect of this process is an increase in energy yield through aerobic respiration.

Although it is clear that cytolysin contributes to virulence in animal models, the conflicting data on its effect on host cells precludes a clear understanding of the mechanisms involved. The synergism exhibited with a surface adhesin–like aggregation substance may be most relevant during biofilm-type infections, where bacterial densities reach a quorum beyond the threshold required for the production and toxic activity of cytolysin. The inclusion of the genes that encode for the cytolysin along with those encoding adhesions, such as aggregation substance and Esp, on the E. faecalis pathogenicity island, may thus be a selected outcome that favors fitness (Shankar, Baghdayan, & Gilmore, 2002).

The molecular mechanism of cytolysin action on erythrocytes has been well studied. Initial studies of the hemolytic activity of the cytolysin revealed that when E. faecalis strains that were hemolytic on blood agar were cultured in standard liquid laboratory media, no such activity could be detected in the culture fluid (Todd, 1934), as noted as a caveat in the above studies with immune cells in culture. These observations prompted the author to term it a “pseudohaemolysin.” Kobayashi (Kobayashi, 1940) later observed that some, but not all, erythrocytes were susceptible to cytolysin-mediated hemolysis; specifically, erythrocytes from human, horse, cow, and rabbit, but not sheep or goat, were susceptible. Later, it was suggested that phosphatidylcholine may serve as the preferred target on the erythrocyte membrane, due to the relatively higher levels of phosphatidylcholine in the outer leaflet of human, horse, rabbit, and mouse erythrocytes (Roelofsen, de Gier, & van Deenen, 1964). Miyazaki et al. (Miyazaki, et al., 1993) provided further support for this contention by demonstrating that phosphatidylcholine was able to inhibit the lysis of horse erythrocytes by hemolytic E. faecalis. By producing non-cytolytic mutants of E. faecalis through repeated exposure to the mutagen nitrosoguanidine, and examining zones of hemolysis on blood agar between pairs of defined mutants, it was determined that the cytolysin consisted of an activator component and a lytic component (Granato & Jackson, 1969).

The cytolysin determinant was first identified on large conjugative plasmids (Clewell, et al., 1982; Dunny & Clewell, 1975; Granato & Jackson, 1969; LeBlanc & Lee, 1982). Localization to conjugative plasmids, which were also found to be responsive to pheromones, was achieved through broth mating experiments (Dunny, Brown, & Clewell, 1978; Dunny, Craig, Carron, & Clewell, 1979). Through transposon mutagenesis and restriction analysis of a prototype pheromone-responsive transmissible plasmid, pAD1, the cytolysin determinant was mapped to a region that spanned 8 kb. The generation of a mutant in this locus allowed investigation into the role of the cytolysin in the pathogenesis of enterococcal infection (Clewell, et al., 1982). Further characterization of cytolysin determinants in different strains revealed its location on either pheromone-responsive plasmids (LeBlanc, Lee, Clewell, & Behnke, 1983), or the chromosome (Ike & Clewell, 1992) within a pathogenicity island (Shankar, Baghdayan, & Gilmore, 2002).

The organization, expression, and regulation of the cytolysin operon have been described in a number of reviews (Coburn & Gilmore, 2003; Cox, Coburn, & Gilmore, 2005; Shankar, Coburn, Pillar, Haas, & Gilmore, 2004; Van Tyne, Martin, & Gilmore, 2013) and are depicted in Figure 2. Briefly, the cytolysin is encoded by a complex operon that consists of eight genes, with expression driven by a pair of divergent, overlapping promoters. Structural genes for the cytolysin subunits (cylLL and cylLS), post-translational modification and secretion functions (cylM, cylB and cylA) and cell immunity (cylI) are transcribed as a single unit. The regulatory genes, cylR1 and cylR2, are transcribed in the opposite direction as a second transcriptional unit. The cytolysin subunits CylLL and CylLS are synthesized as 68- and 63-residue precursors, respectively, and then are post-translationally modified by the product of the third gene (cylM) in the operon (Booth, et al., 1996; Gilmore, et al., 1994). CylM, a 993-residue polypeptide, introduces modifications that are characteristic of the lantibiotic class of bacteriocins. The CylB protein functions as both an ATP-binding cassette transporter (Gilmore, Segarra, & Booth, 1990; Gilmore, et al., 1994) and a signal peptidase (Håvarstein, Diep, & Nes, 1995), and these dual functions serve to export the cytolysin subunits and to proteolytically remove a leader peptide from each subunit. The secreted subunits, which are still inactive at this point, require further protelolytic processing by the CylA protease to acquire activity (CylLSʹʹ and CylLLʹʹ that is capable of lysing target cells (Booth, et al., 1996). CylI provides a self-immunity function by protecting the producer from the bactericidal activity of the cytolysin.

Figure 2. . Model for the expression of the cytolysin in E.

Figure 2.

Model for the expression of the cytolysin in E. faecalis. The toxin subunits LL and LS are ribosomally synthesized in precursor form, then are post-translationally modified by CylM, which introduces modifications characteristic of the lantibiotic class (more...)

CylR1 and CylR2, both of which reduce expression from the cytolysin promoter by about 40-fold, regulate cytolysin production. Purified CylR2 specifically binds to an inverted repeat sequence overlapping the -35 sequence of the cytolysin structural gene promoter (Rumpel, et al., 2004). The crystal structure of CylR2 has revealed dimerization and the presence of a helix-turn-helix DNA binding domain. A model for the action of cytolysin that links sensing of the inducer molecule CylLSʹʹ to the presence or absence of a suitable target cell has been proposed (Coburn, Pillar, Jett, Haas, & Gilmore, 2004). In the absence of a target cell, CylLSʹʹ and CylLLʹʹ are maintained only at basal levels and can further interact to form an insoluble oligomeric complex that effectively diminishes the concentration of free inducer, CylLSʹʹ. Thus, in the absence of a target cell, CylLLʹʹ acts to titrate the level of free CylLSʹʹ in solution to below the threshold level necessary to trigger high-level cytolysin production.

However, in the presence of a target cell, CylLLʹʹ binds preferentially to the target membrane, which reduces the concentration of CylLLʹʹ in solution, and allows the accumulation of free CylLSʹʹ to the level necessary to activate high-level cytolysin expression. Thus, this provides a mechanism by which the bacterium produces high levels of cytolysin only when required by simultaneously detecting both the accumulating inducer CylLSʹʹ molecule and a target cell through the membrane sensor CylLLʹʹ. Furthermore, this would provide an additional fail-safe mechanism to protect the producer from the bactericidal effects of the cytolysin.

Gelatinase, serine protease, and their regulation by Fsr

While bacterial proteases are generally considered to be factors that enhance an organism’s ability to acquire nutrients by proteolysis, they are frequently implicated in damage to host tissues during infections. A broad range of host functions are impacted by bacterial proteases, and include direct or indirect degradation of host connective tissues and proteins (Burns, Jr., Marciel, & Musser, 1996; Lantz, Allen, Duck, Switalski, & Hook, 1991), interference with host cell signaling pathways to facilitate microbial survival (Duesbery, et al., 1998; Maeda & Yamamoto, 1996) and degradation of key components of the host immune system (Plaut, 1983), to name a few. In many cases, they can act as virulence determinants in animal models of infection (Maeda & Yamamoto, 1996).

In E. faecalis, the gelatinase (GelE, a matrix metalloprotease) and a serine protease (SprE) comprise the two secreted proteases that have been well studied (Qin X. , Singh, Weinstock, & Murray, 2001). The term gelatinase was coined for the protease activity based on its observed ability to hydrolyze gelatin (MacCallum & Hastings, 1899). The proteolytic activity, combined with the presence or absence of a hemolytic phenotype, remained the basis for the classification of Streptococcus faecalis into the subspecies S. faecalis, S. faecalis var. liquefaciens, S. faecalis var. hemolyticus, and S. faecalis var. zymogenes—until biochemical and DNA hybridization studies established that S. faecalis and its subspecies liquefaciens and zymogenes were, in fact, the same species, and were later regrouped under the present genus Enterococcus (Farrow, Jones, Phillips, & Collins, 1983). Early reports on the characterization of the gelatinase from E. faecalis suggested that it was a zinc-metalloprotease (Bleiweis & Zimmermann, 1964), and later purification by Makinen et al. (Mäkinen, Clewell, An, & Mäkinen, 1989) from a human oral E faecalis isolate (OG1-10) classified it as a metalloprotease II (EC 3.4.24.4) with broad substrate specificity. The ability of the enzyme to inactivate human endothelin prompted an attempt to rename it as coccolysin, but the term gelatinase has prevailed in the literature (Mäkinen & Mäkinen, 1994). Sequencing of the gelE gene by Su et al. (Su, et al., 1991) showed that it encodes a 509-residue protein predicted to contain a 29 amino-acid signal sequence followed by 162 residues of pro-sequence and a 318 amino-acid mature protein with a mass of 34.5kDa.

Early studies that examined the prevalence of gelatinase production among E. faecalis clinical isolates reported that 72% of hospital isolates were positive (Kühnen, Richter, Richter, & Andries, 1988), while a later study that examined both hospital (clinical and fecal) and community (fecal) isolates noted a higher frequency among hospital isolates (both clinical and fecal), as compared to community fecal isolates. The data suggest a possible enrichment for a Gel+ phenotype in the hospital environment (Coque, Patterson, Steckelberg, & Murray, 1995). The latter study also observed the absence of the Gel+ phenotype in 46% of endocarditis isolates, which suggests no role for this enzyme in this particular disease. Subsequent studies also reported the identification of E. faecalis isolates that carried the gelE gene, but which were phenotypically negative (Eaton & Gasson, 2001; Qin X. , Singh, Weinstock, & Murray, 2000). A 23.9 kb deletion in the region that encodes the fsr genes, which drive gelE expression, was reported among clinical urine isolates of E. faecalis (Nakayama, Kariyama, & Kumon, 2002). A survey of 215 E. faecalis isolates concluded that neither the fsr locus or gelatinase production was enriched in disease-associated isolates, as compared to isolates from healthy individuals (Roberts, Singh, Okhuysen, & Murray, 2004). The majority of gelatinase-negative isolates in this study also exhibited the 23.9 kb deletion previously mentioned. And as noted above, a large prospective study of enterococcal bacteremia patients failed to find a statistically significant correlation between 14-day mortality and gelatinase, cytolysin, or Esp, either singly or in combination (Vergis, et al., 2002).

In well-controlled animal studies that used similar or isogenic strains of E. faecalis and inbred animals, a contribution of gelatinase to the severity of infection is usually shown. Using germ-free rats, Gold et al. (Gold, Jordan, & van Houte, 1975) showed that the protease-positive E. faecalis isolate (OG1) was cariogenic when compared to three other non-proteolytic strains. Further, Gutschik et al. (Gutschik, Møller, & Christensen, 1979) evaluated 10 strains of E. faecalis with varying gelatinase activity in a rabbit endocarditis model, and concluded that the clinical severity of the disease correlated with proteolytic activity. In more controlled studies using isogenic strains, the role of gelatinase has been evaluated in mouse and rat peritonitis models (Dupont, Montravers, Mohler, & Carbon, 1998; Singh, Qin, Weinstock, & Murray, 1998). In both studies using the mouse model, it was noted that a gelatinase-positive strain, OG1RF, exhibited a lower LD50 and an earlier time to death, as compared to a gelatinase-negative strain, OG1X, which was derived from a common parent by nitrosoguanidine mutagenesis. In the rat peritonitis model, however, the OG1X strain induced no mortality and was deemed less pathogenic. Due to the residual gelatinase activity still exhibited by OG1X and the likelihood of other unknown mutations caused by the nitrosoguanidine treatment, a defined insertion mutation in the gelE gene of OG1RF was created (Singh, Qin, Weinstock, & Murray, 1998). The resulting strain had no detectable gelatinase activity, and corroborated earlier observations in the mouse peritonitis model. The gelE disruption mutant was, however, found to abrogate expression of the downstream sprE gene that expressed the second serine protease, which limited the conclusions drawn from the earlier work (Qin X. , Singh, Weinstock, & Murray, 2000). More recent work indicated a potential role for GelE in infection, as well as housekeeping functions (Waters, Antiporta, Murray, & Dunny, 2003). In this study, GelE was shown to limit chain length, clear the bacterial surface of misfolded aggregation substance mutant proteins, enhance autolysis, and reduce the titer of the cCF10 pheromone that induces conjugation of pCF10. While the importance of proteases in biofilm formation through control of extracellular DNA release has been reported (Thomas, Thurlow, Boyle, & Hancock, 2008), others have also shown a role for gelatinase and the Fsr system in facilitating E. faecalis translocation in vitro across polarized human enterocyte-like T84 cells (Zeng, Teng, & Murray, 2005).

Identification of the determinant that encodes the second protease (named SprE, due to its similarity to the S. aureus V8 protease) came about after sequencing the region immediately downstream of gelE in strain OG1-10 (Su, et al., 1991). Qin et al. (Qin X. , Singh, Weinstock, & Murray, 2000) used nucleic acid hybridization with gelE and sprE probes and RT-PCR to demonstrate co-localization and co-transcription of gelE with sprE and the loss of the sprE transcript in the gelE disruption mutant. Also, an OG1RF sprE disruption mutant exhibited gelatinase activity, but not serine protease activity. Further, the sprE deletion mutant resulted in significantly prolonged survival in a mouse peritonitis model, as compared to the wild type OG1RF strain, which implies an independent role for sprE in this model. The attenuated virulence of sprE mutants has also been demonstrated in the C. elegans model (Garsin, et al., 2001; Sifri, et al., 2002) and in the rabbit endophthalmitis model (Engelbert, Mylonakis, Ausubel, Calderwood, & Gilmore, 2004; Suzuki, et al., 2008).

The regulatory aspects of gelatinase and serine protease expression in E. faecalis, as detailed in Figure 3, were unraveled following the identification of the fsr (E. faecalis regulator) locus, a set of three genes (fsrA, fsrB, and fsrC) upstream of gelE (Qin X. , Singh, Weinstock, & Murray, 2000). This locus bears significant similarity to the agr (accessory gene regulator) locus in S. aureus, a quorum-sensing system that positively regulates the expression of several secreted proteins and toxins, and down-regulates the expression of surface proteins (Novick & Geisinger, 2008). Based on sequence homology, FsrA (247 amino acids) and FsrC (447 amino acids) likely constitute a two-component histidine kinase sensor (FsrC) and response regulator (FsrA) system. The FsrB (242 amino acids) protein is similar to AgrB, but has a 50 amino acid carboxy terminal extension, which is processed into an 11-residue peptide pheromone termed the gelatinase biosynthesis-activating pheromone (Nakayama, et al., 2001; Qin X. , Singh, Weinstock, & Murray, 2001).

Figure 3. . Model for the mechanism of fsr activation in E.

Figure 3.

Model for the mechanism of fsr activation in E. faecalis and its effect on the synthesis of gelatinase and serine protease. The E. faecalis fsr system bears global similarity to the staphylococcal agr quorum-sensing system. The secreted gelatinase biosynthesis-activating (more...)

Isogenic E. faecalis OG1RF mutants, with disruptions in the fsrA, fsrB, and fsrC genes, were tested for virulence in a mouse peritonitis model, and all were shown to significantly prolong the time course of survival when compared to the parental strain OG1RF (Qin X. , Singh, Weinstock, & Murray, 2000). In experiments conducted with an fsrB deletion mutant and fsrA, fsrB, and gelE disruption mutants using a C. elegans model, results similar to that in the mouse peritonitis model were seen (Garsin, et al., 2001; Shu & Zhulin, 2002). However, since none of the fsr mutants showed detectable gelatinase or serine protease activity, it was difficult to assign independent role(s) to each, which implies that the observations could have resulted from either polar or regulatory effects of the fsr genes on gelE and sprE expression. Transcriptional and Northern blot analyses provided further insights into the organization of the fsr locus and showed that: (i) disruption of each of the fsr genes abolished a gelE/sprE transcript; (ii) complementation of the fsr locus in trans restored gelatinase activity to each of the three fsr mutants, but not to a gelE disruption mutant; (iii) fsrB and fsrC are co-transcribed; (iv) both fsrB and fsrC are required for fsr locus function; and (v) the Fsr system is autoregulated and functions as a two-component regulatory system (Qin X. , Singh, Weinstock, & Murray, 2001; Qin X. , Singh, Weinstock, & Murray, 2000). Furthermore, primer extension analysis identified promoter sequences upstream of fsrA, fsrB, and gelE, but not fsrC or sprE, and deletions in conserved repeats within these regions abolished promoter activity (Qin X. , Singh, Weinstock, & Murray, 2000).

Experiments analyzing induction of fsrC expression by supernatants from post-exponential and stationary phases of OG1RF and an fsrB deletion mutant suggested that an inducer molecule regulates fsrC expression (Qin X. , Singh, Weinstock, & Murray, 2000). Consistent with these observations, Nakayama et al. (Nakayama, et al., 2001) described the isolation of an 11-aa inducer (pheromone) from late exponential phase cultures and named it gelatinase-biosynthesis activating pheromone (GBAP). The peptide appeared to be derived from residues 220-230 at the carboxy terminus of fsrB. A similar autoinducing peptide lactone (AgrD) of the agr system drives expression of the agr locus in S. aureus (Nakayama, et al., 2001). More recently, a revised model for GBAP synthesis has been proposed, based on the demonstration that GBAP is encoded by a fourth gene, fsrD, within the fsr locus that is separate from fsrB. Even though fsrD is in-frame with fsrB, it is translated independently (Nakayama, et al., 2006). A recent study demonstrated a high degree of conservation of the fsrD gene sequence among multiple MLSTs (multilocus sequence types) in the genome of 22 unrelated E. faecalis strains, although there was considerable variation in the fsr locus outside of fsrD (Galloway-Peña, Bourgogne, Qin, & Murray, 2011).

The effects of mutations in the fsr locus have been examined in a rabbit model of endophthalmitis (Mylonakis, et al., 2002; Engelbert, Mylonakis, Ausubel, Calderwood, & Gilmore, 2004). While an fsrB mutant shown to be defective in the production of both gelatinase and serine protease was attenuated in virulence (Mylonakis, et al., 2002), a later study revealed that the greater degree of attenuation in the fsrB mutant might be due either to synergy or to effects outside of gelatinase and serine protease (Engelbert, Mylonakis, Ausubel, Calderwood, & Gilmore, 2004). Another recent study examined the protease-positive strain OG1S and its isogenic protease-deficient strain OG1X in an aphakic (lacking the eye lens) rabbit model of postoperative endophthalmitis (Suzuki, et al., 2008). The protease-positive bacteria or culture supernatants derived from their in vitro growth in BHI broth decreased ERG b-wave amplitude and caused morphological changes to the posterior capsule and retina (Suzuki, et al., 2008). In a recent study, Singh et al. found that an E. faecalis OG1RF mutant with a nonpolar deletion in fsrB, which produced low levels of gelatinase upon prolonged in vitro incubation, was similar to the wild-type strain in induction of endocarditis in a rat model. In contrast, a gelE insertion mutant was attenuated (Singh K. V., Nallapareddy, Nannini, & Murray, 2005). To further explore if additional genes beyond gelE and sprE are influenced by the fsr locus, Bourgogne et al. (Bourgogne, Hilsenbeck, Dunny, & Murray, 2006) compared the transcription profiles of OG1RF and an isogenic fsrB deletion mutant by microarray analysis. The results revealed that a number of other genes, including those related to biofilms, surface proteins, and metabolic pathways, may be impacted by the fsr locus, and that it may also act as a negative regulator.

Based on the observation that gelatinase destroys a defense system in insect hemolymph (Park, Kim, Lee, Seo, & Lee, 2007), the virulence of fsrB and gelE deletion mutants were recently evaluated in the Galleria mellonella insect model (Gaspar, et al., 2009). When inoculated with wild-type E. faecalis OG1RF or an isogenic fsrB deletion mutant (TX5266), it was observed that 80% of the larvae were dead at 48 hours, as compared to 50% mortality in larvae infected with an isogenic gelE deletion mutant (TX5264), which suggests that only gelE played a role in OG1RF virulence in this model. However, when similar isogenic mutants prepared from two unrelated E. faecalis food isolates were compared in this model, the fsrB deletion mutants were more significantly attenuated than the gelE deletion mutants (Gaspar, et al., 2009).

The Fsr system regulates some of the other virulence determinants discussed in this chapter. For example, regulation of the expression of Ace, an adhesion molecule on the surface of E. faecalis that mediates binding to collagen (see below), was recently described (200). Both fsr and gelE mutants were shown to express significantly higher levels of Ace at the cell surface, and GelE-dependent cleavage of Ace from the surface of E. faecalis was also correlated with improved adhesion to collagen. In a microarray analysis of the Fsr system’s regulon, Bourgogne et al. (Bourgogne, Hilsenbeck, Dunny, & Murray, 2006) described the positive regulation of EbpR, a transcription factor necessary for the production of pili, in addition to other regulators. Negative regulation of the eut locus, which encodes the proteins necessary for the catabolism of ethanolamine, was also noted (Bourgogne, Hilsenbeck, Dunny, & Murray, 2006). As a result of these contributions to the microbe/host dynamic, the Fsr quorum-sensing system has been suggested as a potential target for antimicrobial development. Recent studies that examined culture filtrates of Streptomyces sp. strain Y33-1 identified the known peptide antibiotic siamycin as having a potent inhibitory effect on the production of both gelatinase and GBAP, possibly through a direct interaction with FsrC (Nakayama, et al., 2007).

Adhesins

Aggregation substance

Aggregation substance (AS) proteins are a group of closely related, multifunctional, surface-anchored polypeptides encoded by pheromone-responsive, conjugative plasmids with roles in E. faecalis plasmid transfer and virulence. The AS proteins Asa1, Asc10, and Asp1, encoded by the asa1, prgB, and asp1 genes of conjugative plasmids pAD1, pCF10, and pAD1, respectively, are greater than 90% identical at the amino acid level, outside of a variable region in the N-terminal portion of the protein (Galli, Friesenegger, & Wirth, 1992; Galli, Lottspeich, & Wirth, 1990; Kao, Olmsted, Viksnins, Gallo, & Dunny, 1991). The AS proteins have an N-terminal signal sequence, a C-terminal LPxTG cell wall anchor domain, two Arg-Gly-Asp (RGD) motifs, and a number of experimentally determined domains that were found to mediate aggregation and lipoteichoic acid (LTA)-binding (Hendrickx, Willems, Bonten, & van Schaik, 2009). Expression of AS on the surface of pheromone-induced donor cells leads to strong binding of donors to plasmid-free recipient cells to enhance the efficiency of plasmid transfer by conjugation (194). In addition to its function in bacterial cell aggregation, AS is associated with increased virulence and mortality in rabbit models of infective endocarditis (Chow, et al., 1993; Hirt, Schlievert, & Dunny, 2002; Schlievert, Chuang-Smith, Peterson, Cook, & Dunny, 2010). AS may enhance E. faecalis virulence by serving as an adhesin in bacterial-host interactions, promoting host cell internalization of bacteria, and by contributing to the evasion of killing by phagocytic cells. AS was found to be more prevalent in both clinical and fecal samples in the hospital environment than in samples from the community environment (Coque, Patterson, Steckelberg, & Murray, 1995).

Since many E. faecalis infections appear to originate from bacteria that translocate through the intestinal or genitourinary epithelium (77), E. faecalis must have mechanisms for interaction with host cells and/or extracellular matrix (ECM) proteins. AS has been investigated for this capacity due to its known adherence properties, coupled with the observation that it has dual RGD motifs, which are found in eukaryotic proteins that interact with integrins. AS was first shown to increase the ability of a pAD1-positive strain, relative to a plasmid-free strain, to bind to cultured renal tubular cells (Kreft, Marre, Schramm, & Wirth, 1992). Adherence was disrupted by pre-incubation of the renal cells with synthetic Arg-Gly-Asp-Ser peptides (Kreft, Marre, Schramm, & Wirth, 1992). The N-terminal RGD motif of Asa1 was found to interact with integrin CD11b/CD18 (complement receptor type 3), which led to augmented binding of AS+ cells to macrophages (Süßmuth, et al., 2000); similar results were obtained with polymorphonuclear leukocytes (Vanek, et al., 1999). Enhanced internalization of AS-expressing E. faecalis cells has been demonstrated in HT-29 enterocytes (Olmsted, Dunny, Erlandsen, & Wells, 1994; Sartingen, Rozdzinski, Muscholl-Silberhorn, & Marre, 2000), other intestinal epithelial cell lines (Sartingen, Rozdzinski, Muscholl-Silberhorn, & Marre, 2000), and in colonic mucosa (Isenmann, Schwarz, Rozdzinski, Marre, & Beger, 2000). The presence of surface-expressed AS substantially increased macrophage phagocytosis of E. faecalis cells. The bacteria subsequently suppressed the macrophage respiratory burst and were resistant to intracellular killing (Süßmuth, et al., 2000). Similarly, AS augmented opsonization-independent phagocytosis, yet the bacteria were resistant to intracellular killing in activated human PMNs (Rakita, et al., 1999). Asa1 was also shown to significantly enhance binding to the ECM components fibronectin, thrombospondin, vitronectin, and type I collagen, but not to laminin or type IV collagen (Rozdzinski, Marre, Susa, Wirth, & Muscholl-Silberhorn, 2001). The particularly strong interaction between Asa1 and fibronectin was dependent on an intact variable region within the N-terminal half of the protein (Rozdzinski, Marre, Susa, Wirth, & Muscholl-Silberhorn, 2001).

A significant effort has been undertaken to dissect the functional domains of pCF10’s Asc10, with respect to aggregation, host cell binding, and endocarditis virulence. Using a library of insertional mutants distributed throughout prgB, Waters and Dunny (Waters & Dunny, 2001) defined an aggregation domain in the N-terminal half of the protein (amino acids 473-683) that included the first RGD motif and overlapped part of the variable region. They further showed that an intact aggregation domain was required for the efficient uptake by HT-29 cells through an unknown mechanism that was independent of the RGD motifs. Aggregation itself was not a prerequisite for uptake (Waters, Wells, & Dunny, 2003). Site-specific mutations in the RGD motifs also had no effect on aggregation (Waters, Wells, & Dunny, 2003). In experiments that studied the direct interaction of purified Asc10 variants with LTA, Waters et al. (Waters, et al., 2004) discovered that the LTA-binding domain is a separate domain situated upstream of the Asc10 (473-684) aggregation domain, at amino acids 150-358, and that this domain was also required for aggregation and internalization by HT-29 enterocytes. Recent advances in enterococcal genetic manipulation techniques (Kristich, Chandler, & Dunny, 2007) have facilitated the construction of a markerless in-frame prgB deletion in pCF10, as well as the generation of pCF10 variants carrying mutant prgB alleles that have deletions or point mutations in the LTA-binding aggregation domain and the RGD motifs (Chuang, et al., 2009). When evaluated in a rabbit experimental endocarditis model, the pCF10ΔprgB strain and a strain expressing a full-length prgB variant with glycine-to-alanine point mutations in both RGD motifs (RGD double mutant) showed the most pronounced decreases in virulence (Chuang, et al., 2009). Mutation of either RGD motif alone revealed that the N-terminal RGD motif was more important in virulence. In fact, a strain with a 450-amino acid C-terminal region deletion, which encompassed the second RGD domain, remained fully virulent. Disruption of either aggregation domain resulted in intermediate levels of attenuation. In an ex vivo porcine heart valve model that evaluated bacterial adherence at early time points, the pCF10ΔprgB strain was found to colonize valve tissues at only 40% of the level of the Asc10+ strain after four hours (Chuang-Smith, Wells, Henry-Stanley, & Dunny, 2010). Mutations in either or both aggregation domains, or both RGD motifs, caused a slight impairment in valve colonization, whereas deletion of the C-terminal subdomain did not cause any significant defects (Chuang-Smith, Wells, Henry-Stanley, & Dunny, 2010). The authors propose separate virulence functions for the multiple domains—the aggregation domains likely mediate binding at the heart valve surface, whereas it was speculated that the RGD motifs may contribute to immune evasion (Chuang, et al., 2009; Chuang-Smith, Wells, Henry-Stanley, & Dunny, 2010). In heart valve infections caused by AS+ enterococci, heart valve colonization may be accelerated by the ability of interbacterial aggregates to attach to the host surface as a single event (Chuang-Smith, Wells, Henry-Stanley, & Dunny, 2010).

As noted above, the combination of AS and cytolysin resulted in increased virulence in experimental endocarditis and a mouse intraperitoneal injection model (Chow, et al., 1993; Dupont, Montravers, Mohler, & Carbon, 1998). However, when tested in other infection models, the presence of AS did not enhance the killing of C. elegans (Garsin, et al., 2001) or contribute to pathogenesis in endophthalmitis or ascending unobstructed urinary tract infections (Jett, Atkuri, & Gilmore, 1998; Johnson, Clabots, Hirt, Waters, & Dunny, 2004). This may be partially due to the failure of AS induction in vivo (Johnson, Clabots, Hirt, Waters, & Dunny, 2004).

In the course of infection, AS can be induced in the absence of recipient cells or the addition of an exogenous pheromone. This was first observed in tissue culture experiments and was found to be caused by a component found in serum and plasma (Hirt, Schlievert, & Dunny, 2002; Kreft, Marre, Schramm, & Wirth, 1992). The prevention of autocrine induction of Asc10 on pCF10 (by self-produced pheromone) is a tightly regulated process that involves an 80:1 molar ratio of the inhibitor peptide, iCF10, to the activating peptide, cCF10 (Nakayama, Dunny, Clewell, & Suzuki, 1995). Genetic and functional evidence supports a model for Asc10 self-activation in vivo that occurs in response to disruption of the inhibitor:activator peptide ratio, which follows the inactivation of iCF10 by a component in plasma. The component is possibly albumin or albumin/lipid complexes (Chandler, Hirt, & Dunny, 2005). Induction of Asc10 in endocarditis (Hirt, Schlievert, & Dunny, 2002) is supported by data that demonstrate that pCF10ΔprgB leads to significant attenuation in this model (Chuang, et al., 2009). Despite a demonstrable role for aggregation substance in enterococcal endocarditis, the vaccination of rabbits with a purified surface-exposed region of Asc10 failed to provide protection (McCormick, et al., 2001). Asc10 antibodies were excluded from vegetations in passively immunized animals, and opsonized bacteria in animals pre-immunized with Asc10 were still found in vegetations (McCormick, Tripp, Dunny, & Schlievert, 2002). Unexpectedly, immunization with heat- and gentamicin-killed AS+ organisms increased severity of infection when immunized animals were challenged with the AS+ organism, as compared to animals vaccinated with an AS- strain (Schlievert, Chuang-Smith, Peterson, Cook, & Dunny, 2010). However, passive immunization with anti-AS IgG Fab significantly reduced endocarditis disease that was caused by an AS+ strain of E. faecalis (Schlievert, Chuang-Smith, Peterson, Cook, & Dunny, 2010).

Enterococcal surface protein, Esp

Enterococcal surface protein, or Esp, was identified initially in a highly virulent, gentamicin-resistant, E. faecalis isolate from a bacteremia (Shankar V. , Baghdayan, Huycke, Lindahl, & Gilmore, 1999). This ~202 kDa protein possesses structural features that are characteristic of Gram-positive surface proteins, such as a transport signal sequence and a cell wall-anchor sequence that is slightly divergent from the consensus LPxTG motif. The core region consists of repeat units that make up about 50% of the protein, and has a unique architecture made up of distinct tandem repeating units. The first repeating unit located downstream of the 694 amino acid N-terminal domain consists of three 84-residue repeats that are specified by nearly identical 252-nucleotide tandem repeats (A repeats). Seven, nearly identical, 246-nucleotide tandem repeating units (C repeats), which encode reiterations of an 82-amino acid sequence, are flanked by the B repeats, which share 74% sequence identity at the amino acid level. These highly conserved, multiple repeat structures allow for expression of alternate forms that differ in the number of repeat units as a result of recombination mediated addition or deletion of units (Shankar V. , Baghdayan, Huycke, Lindahl, & Gilmore, 1999). Esp exhibits global structural similarity to the Streptococcus pyogenes protein R28 (Stålhammar-Carlemalm, Areschoug, Larsson, & Lindahl, 1999), Streptococcus agalactiae Rib and C-alpha proteins (Michel, Madoff, Kling, Kasper, & Ausubel, 1991) and the Staphylococcus aureus biofilm-associated protein, Bap (Cucarella, et al., 2001). This similarity is restricted to a highly conserved region within the C repeat units of the Esp protein, corresponding to regions within the group A and B streptococcal proteins, while the similarity with Bap is limited to the non-repeat N-terminal region.

The esp gene was found to be enriched in infection-derived E. faecalis isolates (Shankar V. , Baghdayan, Huycke, Lindahl, & Gilmore, 1999), and an esp homolog was reported in E. faecium isolates (Baldassarri, et al., 2001; Willems, et al., 2001). The variant esp gene was significantly enriched (P < 0.0001) among epidemic vancomycin-resistant E. faecium isolates (VREF) that are genetically distinct from non-epidemic VREF obtained from hospitals on three continents (Willems, et al., 2001). A comparison of 22 vancomycin-resistant E. faecium isolates from catheter-related, bloodstream infections (VREF-CRB) to 30 VREF isolates from the gastrointestinal tract of control patients resulted in no correlation between esp in VREF and bacteremia, or with more biofilm formation (Raad, et al., 2005). A study in the United Kingdom detected esp in over 60% of vancomycin-resistant and vancomycin-sensitive clinical isolates, but not in environmental isolates (Woodford, Soltani, & Hardy, 2001). Although no correlation with vancomycin resistance was observed, another survey of 201 clinical bloodstream isolates found esp to be present in 52% of E. faecium, 40% of E. faecalis, and one E. raffinosus strain (88). A screening study of enterococcal virulence factors also identified the esp homolog to be enriched among clinical E. faecium isolates, as compared with food or starter isolates (Eaton & Gasson, 2001). The esp gene was found to be significantly associated with ampicillin-resistant strains, as compared to ampicillin-sensitive strains of E. faecium (P < 0.001), regardless of the isolation site (Coque, Willems, Cantón, Del Campo, & Baquero, 2002). However, as noted above, one enterococcal bacteremia study did not find a statistically significant correlation between 14-day mortality and gelatinase, cytolysin, or Esp, either singly or in combination (Vergis, et al., 2002).

Genotyping of endodontic enterococcal isolates showed that 20 of 31 E. faecalis and 2 of 2 E. faecium isolates carried the esp gene (Sedgley, et al., 2005). Seno et al. (Seno, Kariyama, Mistuhata, Monden, & Kumon, 2005) examined 352 E. faecalis isolates from patients with complicated urinary tract infections and found that 75% of the strains carried the esp gene. Isolates carrying both esp and asa1 genes (315/352) were found to be better biofilm formers (P = 0.038) than isolates that carried neither gene. In a study of E. faecium clinical isolates from Germany, the esp gene was found among multiple MLST types belonging to clonal complex 17 (CC-17) (Klare, et al., 2005), in which Esp was later described as being an important biofilm determinant (Heikens, Bonten, & Willems, 2007). A recent study in Australia of 41 VREF vanB isolates from immunocompromised patients (n= 41; 14 infected and 27 colonized) found the esp gene was highly prevalent, but not associated with 30-day mortality (Worth, et al., 2008). Another study in India found the esp gene to be significantly enriched among 200 infection-derived isolates of E. faecalis from patients at a tertiary care center, as compared to 100 commensal isolates. The presence of the gene was highly correlated with biofilm formation in vitro (Upadhyaya, Lingadevaru, & Lingegowda, 2011).

Toledo-Arana et al. (Toledo-Arana, et al., 2001) found a significant correlation between the presence of esp and the ability of E. faecalis to form biofilms on polystyrene. Ninety-three percent of the tested esp-positive isolates formed a biofilm, as compared to none of the esp-negative isolates. The presence of esp did not, however, promote the adhesion of E. faecalis to other medically relevant substrates, such as silicone rubber, fluoroethylene-propylene, or polyethylene (Waar, van der Mei, Harmsen, Degener, & Busscher, 2002). In evaluating the adhesion of enterococcal strains to two types of urinary catheter materials, Joyanes et al. (Joyanes, Pascual, Martínez-Martínez, Hevia, & Perea, 2000) concluded that E. faecalis showed greater adherence than E. faecium. Furthermore, adherence was not related to either bacterial surface hydrophobicity or hemolysin or gelatinase production. An investigation of adhesive properties of E. faecalis strains to intestinal Int-407 and Girardi heart cell lines revealed no role for Esp in adhesion (Archimbaud, et al., 2002). Infection-derived E. faecium isolates that were positive for esp were reported to adhere better to Caco-2 cells than esp-negative isolates (P < 0.05), but these studies were done with non-isogenic strains (Lund & Edlund, 2003). In a later study, these authors also showed that esp-positive, infection-derived E. faecium exhibited higher conjugation frequencies (P < 0.01) with respect to acquisition of vanA, as compared to esp-negative isolates (Lund, Billström, & Edlund, 2006). The esp gene was also found to be present in a large number of antibiotic resistant, cross-transmitted, E. faecium isolates prevalent in Swedish hospitals (Billström, Sullivan, & Lund, 2008).

The role of Esp in colonization and persistence of E. faecalis in an animal model of ascending urinary tract infection was evaluated by comparing an Esp-positive strain of E. faecalis to its isogenic Esp-deficient mutant (Shankar, et al., 2001). Groups of CBA/J mice were challenged transurethrally with 108 CFU of either the parent or the mutant strain, and bacteria were enumerated in the urine, bladder, and kidneys five days post-infection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the Esp-bearing parent strain than from mice challenged with the Esp-deficient mutant, which points to a role for Esp as a virulence factor in this infection model. The study suggested that Esp may serve to promote bacterial adhesion to the bladder epithelium through specific components of the bladder wall, such as mucin or uroplakin (Shankar, et al., 2001).

The role of esp in experimental peritonitis and urinary tract infection has been recently evaluated using E. faecium E1162 and an isogenic esp-deficient mutant (Leendertse, et al., 2009). Results from this study corroborated observations made for E. faecalis and revealed enhanced binding of the wild type strain to bladder and kidney epithelial cells in vitro, as well as higher numbers in both organs among infected mice. No difference was observed in the peritonitis model. The same isogenic pair was recently tested in a mouse bacteremia model (Sava, et al., 2010). In this study, passive immunization with antibodies to the N-terminal portion of Esp did not protect mice from bacteremia (P > 0.05), while in comparison, antibodies to LTA from both E. faecalis and E. faecium resulted in fewer numbers of E. faecium in the blood.

The role of E. faecium Esp in a rat model of infectious endocarditis was recently reported (Heikens, et al., 2011). In this study, higher numbers of the Esp-positive strain (E1162) were recovered at 24 hours from heart vegetations, as compared to an isogenic, Esp-deficient mutant. Further, anti-Esp antibodies were detected in sera of infected mice and from infected patients. In similar studies, the esp-positive strain MMH594 was recovered in significantly higher numbers (P < 0.01) from 48-hour vegetations on infected heart valves in a rabbit endocarditis model, as compared to an isogenic, esp-deficient mutant (unpublished). Although the presence of antibodies to Esp was not evaluated in this study, the presence of Esp appeared to significantly enhance vegetation weight.

Ace, an adhesin to collagen of E. faecalis

Attachment of bacteria to host tissue components, such as the extracellular matrix (ECM), is an important early step of the infection process. Several studies have reported the ability of some E. faecalis isolates to adhere to a number of extracellular matrix (ECM) proteins, such as collagen, laminin, fibrinogen, fibronectin, lactoferrin, vitronectin, and thrombospondin, but most of them agree that adherence to these proteins is exhibited by relatively few isolates after growth in standard laboratory media (233, 291, 298). For example, Xiao et al. (Xiao, Höök, & Weinstock, 1998) found that only 2 of 44 E. faecalis isolates adhered to collagen and/or laminin after growth in brainheart infusion broth (BHI) at 37°C; but growth at an elevated temperature of 46°C led to a significant increase in the adherence of most clinical E. faecalis isolates to collagen and laminin, but not to fibronectin, fibrinogen, or albumin. Similar conditional adherence to collagen, fibrinogen, and fibronectin was later found after growth of E. faecalis in the presence of serum, versus its growth in BHI (180). Of note, a brief exposure (<5 min) to serum caused an immediate increase in adherence to fibronectin and collagen, to a lesser extent, while growth in the presence of serum was required for fibrinogen adherence (Nallapareddy & Murray, 2008). Thus, these observations suggest that adherence phenotypes to ECM proteins are not constitutively expressed by most clinical E. faecalis isolates, but are instead elicited by a stress condition or a host-derived cue, or are mediated by a potential factor(s) in serum-forming bridges between bacterial adhesins and host ECM proteins.

Subsequent searches for genes that encode potential adhesins in the first available genome sequence of E. faecalis (strain V583) led to the discovery of Ace (adhesin to collagen of E. faecalis), an MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein with similarity to the ligand-binding region of the S. aureus collagen adhesin Cna. A recombinant version of Ace was shown to bind to collagen type I, an important fibrillar collagen type with a wide tissue distribution, although with different kinetics than Cna (Rich, et al., 1999). Unlike Cna, however, Ace was also found to bind to collagen type IV and laminin, both of which are major network-forming components of mammalian basement membranes (Nallapareddy, Qin, Weinstock, Höök, & Murray, 2000). Using an isogenic ace mutant of E. faecalis strain OG1RF, Nallapareddy et al. (Nallapareddy, Qin, Weinstock, Höök, & Murray, 2000) further demonstrated that native Ace displayed on the cell surface mediates the majority of the 46°C-elicited collagen and laminin adherence of OG1RF. Direct binding of native Ace to collagen was confirmed by a far-western analysis of mutanolysin extracts from 46°C-grown OG1RF cells. Rabbit antibodies specific to the collagen-binding A-domain of Ace almost completely inhibited the conditional collagen and laminin adherence of OG1RF, as well as two other clinical E. faecalis strains, which further corroborates the specificity of the Ace-ligand interaction (Nallapareddy, Qin, Weinstock, Höök, & Murray, 2000). Similar inhibition was also seen with human-derived antibodies purified from serum of an endocarditis patient (Nallapareddy S. R., Singh, Duh, Weinstock, & Murray, 2000). Ace has also been shown to be important for attachment of E. faecalis to dental roots (Hubble, Hatton, Nallapareddy, Murray, & Gillespie, 2003). Further studies identified the collagen-rich dentin as the target for this Ace-mediated adherence, which suggests that Ace may have a role in the common isolation of E. faecalis from periapical periodontitis, as well as its ability to persist in the root canal in the presence of antibacterial medications (Kowalski, et al., 2006). In addition, Hall et al. (Hall, et al., 2007) reported that Ace binds to collagen type VI as a recombinant protein, and that fluorescent beads coated with rAce A-domain bind to cultured human epithelial and endothelial cells. However, the moiety(ies) interacting with Ace on these cells remains uncharacterized.

Studies of ace expression revealed much higher levels of ace-specific mRNA after growth in the presence of serum or collagen type IV than in a routine laboratory medium (BHI) (Nallapareddy & Murray, 2006; Shepard & Gilmore, 2002). The increased mRNA was correlated with abundant surface display of Ace on E. faecalis OG1RF and its increased collagen and laminin adherence (Nallapareddy & Murray, 2006). Similar upregulation of ace expression after exposure to collagen was also seen with other E. faecalis strains of different origins, which suggests that this may be a common programmed response of this species to a host-derived signal (Nallapareddy & Murray, 2006). The detection of anti-Ace antibodies in 90% of serum samples from patients with E. faecalis endocarditis suggested that most, if not all, strains produce Ace during infection and that Ace is antigenic in humans (Nallapareddy S. R., Singh, Duh, Weinstock, & Murray, 2000). A recent flow cytometry analysis of bacteria harvested from endocarditis vegetations infected with E. faecalis found Ace on 40-45% of bacterium-size particles, which confirmed the production of Ace in vivo (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010). While details of regulatory pathways that control ace expression are largely unknown, the transcriptional regulator Ers has been shown to negatively regulate ace by binding to its promoter region (Lebreton, et al., 2009). This study also indicated bile salts as another stress/factor that increases production of ace mRNA. Although the molecular details remain uncharacterized, this bile salts effect was proposed to be mediated by the deregulation of ers (Lebreton, et al., 2009). More recently, Pinkston et al. (Pinkston, et al., 2011) reported that gelatinase specifically cleaves Ace from the cell surface in later stages of in vitro growth, which provides at least one explanation for the previous detection of low levels of surface Ace and collagen adherence under these conditions. Using an fsrB mutant, this study further showed that surface display of Ace is modulated by the Fsr system through the activity of gelatinase, which demonstrates a link between Ace-mediated collagen adherence of E. faecalis and the multi-target Fsr quorum-sensing regulatory network (Bourgogne, Hilsenbeck, Dunny, & Murray, 2006; Pinkston, et al., 2011).

Several studies have implicated an important role for Ace in virulence. Using a mouse model of septic arthritis, Xu et al. (Xu, Rivas, Brown, Liang, & Höök, 2004) showed that exogenous expression of the collagen-binding A-domain of Ace in S. aureus increased the virulence of this organism to levels similar to an isogenic S. aureus strain that expressed Cna. Evidence for the role of Ace in virulence of E. faecalis has been shown by attenuation of an ace deletion mutant in a mouse UTI model, a G. mellonella insect model, and in survival within murine peritoneal macrophages (Lebreton, et al., 2009). Singh et al. (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010) recently demonstrated the importance of Ace for E. faecalis pathogenesis in endocarditis by showing that the deletion of ace leads to significant attenuation in experimental rat endocarditis, while no difference was observed in a peritonitis model. Furthermore, Ace was shown to be important in the early attachment stage of endocarditis, in addition to being expressed on the surface of E. faecalis cells in vivo within vegetations—a finding that is consistent with the concept that Ace mediates attachment of E. faecalis to exposed collagen and/or laminin at sites of valvular damage. Finally, vaccination with a recombinant collagen-binding A-domain of Ace, as well as passive immunization with anti-Ace A-domain antibodies, both conferred protection against endocarditis and reduced E. faecalis colonization of vegetations in the rat model (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010). This is in agreement with the previously shown efficient inhibition of collagen and/or laminin adherence by both polyclonal and monoclonal anti-Ace A-domain antibodies in vitro (Hall, et al., 2007; Nallapareddy, Qin, Weinstock, Höök, & Murray, 2000; Nallapareddy S. R., Singh, Duh, Weinstock, & Murray, 2000). These results suggest that Ace, which was also found to be immunogenic in humans (Nallapareddy, Qin, Weinstock, Höök, & Murray, 2000), could be a useful target for immunoprophylactic or therapeutic strategies (Singh K. V., Nallapareddy, Sillanpää, & Murray, 2010).

EfaA, E. faecalis antigen A

EfaA is a major surface antigen of E. faecalis and was identified using sera from patients with known E. faecalis endocarditis (Lowe, Lambert, & Smith, 1995). The efaA gene is the third gene of a three-gene operon, efaBCA, which is predicted to encode components of an ABC-type transporter, with EfaA as its putative substrate-binding lipoprotein component (Low, Jakubovics, Flatman, Jenkinson, & Smith, 2003). EfaA has sequence identity with a group of streptococcal ABC transporter proteins, some of which have also been identified as prominent surface adhesins and/or as factors associated with adherence or pathogenesis (Andersen, Ganeshkumar, & Kolenbrander, 1993; Kolenbrander, Andersen, Baker, & Jenkinson, 1998; Viscount, Munro, Burnette-Curley, Peterson, & Macrina, 1997). Evidence for the previously implied importance of efaA in E. faecalis infection was provided by Singh et al. (Singh, Coque, Weinstock, & Murray, 1998), who showed that mice infected with an isogenic efaA disruption mutant of E. faecalis OG1RF had significantly prolonged survival, as compared to those infected with the wild-type parent strain, in an experimental peritonitis model. Screening for the prevalence of the efaA gene found it to be ubiquitously present in nearly all E. faecalis isolates and identified an equally prevalent homolog in E. faecium (Eaton & Gasson, 2001; Singh, Coque, Weinstock, & Murray, 1998). Low et al. (Low, Jakubovics, Flatman, Jenkinson, & Smith, 2003) reported that EfaA production is up-regulated in vitro when environmental Mn2+ concentrations are low. They proposed that the efaBCA operon encodes a high-affinity manganese permease that is expressed in tissues or serum where Mn2+, an important micronutrient for E. faecalis, is not freely available, which explains the importance of EfaA for the infection of human host tissues (Low, Jakubovics, Flatman, Jenkinson, & Smith, 2003).

Ebp, endocarditis, and biofilm-associated pili

Although filamentous structures resembling pili or fimbriae were seen in electron microscopy studies of enterococci by Handley and Jacob in the 1980s (Handley & Jacob, 1981), the genetic and structural basis of enterococcal pili remained unknown until the discovery of a three-gene ebpABC (endocarditis and biofilm-associated pili) locus and an adjacent downstream sortase-encoding gene, bps (biofilm and pilus-associated sortase), that are necessary for the assembly of pili on the surface of E. faecalis strain OG1RF (Nallapareddy S. R., et al., 2006). The first clue to the importance of the Ebp pili for enterococcal virulence came from the finding of high titers of antibodies against recombinant proteins corresponding to the three structural pilus subunits, EbpA, EbpB, and EbpC, in sera from patients with E. faecalis endocarditis, a finding that implies that they are expressed in vivo and are immunogenic in the human host (Sillanpää, Xu, Nallapareddy, Murray, & Höök, 2004). Nallapareddy et al. (Nallapareddy S. R., et al., 2006) then showed that a non-piliated ebp allelic replacement mutant was significantly attenuated in a rat endocarditis model, and that this mutant was also impaired in its ability to attach to plastic surfaces and form biofilm. In addition, further studies found the ebp locus to be important for the colonization of kidneys in a murine model of ascending UTI (Singh, Nallapareddy, & Murray, 2007). A bps sortase deletion mutant of OG1RF that is unable to polymerize Ebp pilins on the cell surface was similarly attenuated in experimental UTI, and had a decreased ability to form biofilm (Kemp, Singh, Nallapareddy, & Murray, 2007). These studies have identified a role for Ebp pili of E. faecalis in two clinically important infections, in which the ability of E. faecalis to form biofilm is considered to play a major role.

As discussed above, the adherence of most E. faecalis isolates to several host ECM proteins is enhanced by growth in the presence of serum. Recently, deletion of the ebpABC genes was shown to nearly eliminate serum-elicited adherence of E. faecalis OG1RF to fibrinogen, a major serum and ECM component (Nallapareddy S. R., Singh, Sillanpää, Zhao, & Murray, 2011), while no effect on fibronectin adherence was observed. The involvement of Ebp pili in fibrinogen adherence was confirmed by complementation and inhibition of fibrinogen adherence of OG1RF by antibodies against the three Ebp pilins; the latter results also pointed to the possibility of using anti-Ebp antibodies for immunization against E. faecalis infections. Comparison of single and double deletion mutants of ebpABC and ace and their complemented derivatives showed that Ebp pili are also involved in serum-elicited adherence of E. faecalis OG1RF to collagen, although to a lesser degree than Ace. These mutants also showed reduced adherence to a collagen-secreting fibroblast cell line 3T6 (Nallapareddy S. R., Singh, Sillanpää, Zhao, & Murray, 2011). Furthermore, native pili extracted from OG1RF cells were found to bind to collagen, but not fibronectin, which gives further support for a role of Ebp pili in collagen adherence (Nallapareddy S. R., Singh, Sillanpää, Zhao, & Murray, 2011). In contrast, the ebpABC deletion had no significant effect on adherence to two other cell lines—human intestinal (Caco-2) and urinary bladder (T24) epithelial cells—nor on translocation across a human intestinal epithelial (T84) monolayer. This suggests that Ebp pili, unlike pili from streptococci, either do not have a major role in adherence to host cells/translocation or that the cell lines studied do not express the necessary surface receptors/factors.

Bacterial interactions with platelets are known to contribute to colonization of vegetations on heart valves during endocarditis (Fitzgerald, Foster, & Cox, 2006; Moreillon, Que, & Bayer, 2002). Studies with enterococci have shown variable platelet adherence and aggregation properties among isolates (Rasmussen, Johansson, Söbirk, Mörgelin, & Shannon, 2010; Scheld, Zak, Vosbeck, & Sande, 1981; Usui, Ichiman, Suganuma, & Yoshida, 1991). Nallapareddy et al. (Nallapareddy S. R., et al., 2011) recently reported that growth in the presence of serum increases adherence of E. faecalis OG1RF to human platelets. In the same study, using the pilus-deficient ebpABC deletion mutant and its complemented derivative, Ebp pili were shown to mediate the majority of this serum-elicited platelet adherence, while ace and fss2, a gene that encodes a fibrinogen-binding MSCRAMM protein (Sillanpää J. , et al., 2009), had no effect (Nallapareddy S. R., et al., 2011). Consistent with these findings, a pilus negative bps deletion mutant, as well as a housekeeping sortase (srtA) mutant and a sortase double mutant, were also found to be impaired in adherence to platelets (Nallapareddy S. R., et al., 2011).

Studies on regulation of the ebp locus identified a gene designated ebpR (endocarditis- and biofilm-associated pilus regulator), that is immediately upstream of the ebpABC genes and oriented in the opposite direction (Bourgogne, et al., 2007). The EbpR protein was found to have similarity to the AtxA/Mga family of regulator proteins and contains two predicted helix-turn-helix DNA-binding domains (Bourgogne, et al., 2007). Deletion of ebpR led to a 100-fold reduction in the expression of the ebpABC genes (Bourgogne, et al., 2007), which were previously shown to be co-transcribed as an operon (Nallapareddy S. R., et al., 2006). Only a slight effect was seen on bps, which is likely explained by its independent transcription from a second promoter, in addition to a four-gene ebpABC–bps transcript.

Although the regulatory pathway(s) involved in Ebp pilus production largely remain to be elucidated, several studies have shown that production of Ebp pili is affected by environmental factors. These include increased pilus expression when E. faecalis OG1RF is grown in tryptic soy broth + glucose (TSBG; standard biofilm medium) versus BHI, and even greater expression with growth in the presence of serum or bicarbonate (see below) (Bourgogne, et al., 2007; Bourgogne, Thomson, & Murray, 2010; Nallapareddy S. R., et al., 2006). Analysis of a large collection of diverse E. faecalis isolates demonstrated that serum-elicited Ebp pilus production is a general property exhibited by all 91 isolates tested (Nallapareddy S. R., et al., 2011). Further studies by flow cytometry revealed two distinct populations in terms of Ebp surface expression, which suggests a bistable mode of expression similar to recent reports with S. pneumoniae (Basset, et al., 2011; De Angelis, et al., 2011). Even more enhanced Ebp pilus expression was seen when OG1RF cells were harvested directly from endocarditis vegetations; ~70% of bacterial size particles that reacted with anti-E. faecalis whole-cell antibodies were found to produce EbpC compared to ~30% of serum-grown OG1RF, which demonstrates that Ebp pili are actively generated within host vegetations during endocarditis. These results corroborate the previous studies that indicate in vivo Ebp pilus expression by the common finding of anti-Ebp antibodies in sera from endocarditis patients infected with E. faecalis (Sillanpää, Xu, Nallapareddy, Murray, & Höök, 2004).

As mentioned above, bicarbonate,which is another environmental factor that may mimic physiologic conditions in the host, was recently demonstrated to induce Ebp pilus production via EbpR, a positive regulator of the ebpABC operon (Bourgogne, Thomson, & Murray, 2010). Although many other regulators of the AtxA/Mga family and virulence factors of other bacteria are known to be activated by the presence of CO2 or CO2/HCO3-, the increase in ebpR and ebpABC transcripts was found to be caused by the addition of HCO3- and not CO2 (Bourgogne, Thomson, & Murray, 2010). Apart from the increased expression of cytolysin when E. faecalis cells are grown in an atmosphere of 80% H2/20% CO2 (41), Ebp pili were described as the first virulence-associated factor of E. faecalis whose expression is activated in response to elevated levels of bicarbonate. It was postulated that the release of HCO3- in the upper intestinal tract in response to the acidic discharges from the stomach may be sensed by E. faecalis and consequently lead to increased Ebp pilus expression to favor intestinal colonization. The same study also showed that the previously identified role of the Fsr quorum-sensing system as a weak repressor of the ebp locus (Bourgogne, Hilsenbeck, Dunny, & Murray, 2006) is independent of the ebpR-mediated bicarbonate effect. A recent report by Gao et al. (Gao, et al., 2010) identified a previously uncharacterized gene, rnjB, which encodes a putative RNase J2, as another activator of the ebp operon. The ebpABC mRNA levels were significantly lower in an rnjB deletion mutant compared to the wild-type parent strain OG1RF, which suggests that rnjB regulates Ebp pilus expression at the mRNA level. Of note, both the ebpR and rnjB mutants showed lower biofilm formation versus the parent strain, which is consistent with the reduced Ebp pilus production. Considering the findings with ebpR, rnjB and fsr, it appears that several pathways with multiple target genes control the production of Ebp pili. However, the molecular details of these potential regulatory networks are still emerging.

Taken together, the above studies have revealed a multifunctional role for Ebp pili of E. faecalis in host-pathogen interactions. It could be argued that these properties enable circulating E. faecalis cells to adhere to exposed ECM proteins, such as collagen, on damaged vascular endocardial surfaces and to platelets and fibrinogen in vegetations on heart valves, which potentially explains the importance of Ebp pili in experimental endocarditis. Further studies are needed to determine which of the individual Ebp pilins is/are involved in these processes. Similar virulence-associated properties, such as adherence to various host tissue components, biofilm formation, and a role in animal infection models have also been assigned to pili or pilins of an increasing number of other Gram-positive bacteria, including E. faecium (Sillanpää J. , et al., 2010), S. pyogenes (Abbott, et al., 2007), S. agalactiae (Maisey, Hensler, Nizet, & Doran, 2007; Maisey, et al., 2008), S. pneumoniae (Barocchi, et al., 2006; Gianfaldoni, et al., 2007), and C. diphtheriae (Mandlik, Swierczynski, Das, & Ton-That, 2007), which points to a widespread role of these structures in infections caused by Gram-positive bacteria.

Enterococcal Capsule, Lipoteichoic Acid and Cell Wall Polysaccharide

Detailed information about the structure, chemistry, and genetics that underlie the carbohydrate and teichoic acid components of the enterococcal cell wall are presented in Enterococcal cell wall components and structures. The focus of this section will be on the role that some of these components play in enterococcal pathogenesis. Generally, these non-protein components on the outer surface of the bacterium can influence how well the host recognizes the invading pathogen, which affects the efficiency of the mechanisms of clearance, including phagocytosis and/or cytokine production (Hancock & Gilmore, 2002; Huebner, Quaas, Krueger, Goldmann, & Pier, 2000; Theilacker, et al., 2011; Thurlow R. L., Thomas, Fleming, & Hancock, 2009). The cell wall components that enhance the ability of the bacterium to overcome various host defenses are considered to be virulence factors.

The polysaccharide antigenic components of the Gram-positive cell wall can be divided into two categories: those that are common to all members of a given species and those that vary within a species in terms of their composition and/or structure. Antibodies can be raised against components that are surface-exposed, and typing schemes to differentiate enterococcal strains have been developed, based on whole-cell agglutination with particular antibodies (Hufnagel, et al., 2004; Maekawa, Yoshioka, & Kumamoto, 1992; Sharpe, 1964). These antibodies do not simply indicate differences between polysaccharides, which have been shown to vary, but can also indicate whether a static component of the cell wall is exposed or not, due to the variable presence of a capsule in E. faecalis strains (Hufnagel, Carey, Baldassarri, Reinert, & Huebner, 2006; Thurlow, Thomas, & Hancock, 2009). As detailed below, the presence of a capsule generally suggests a more virulent strain because of the immune evasion capabilities that the capsule imparts.

Variable capsular carbohydrate

Several studies have demonstrated that an important method of immune clearance of enterococci is neutrophil-mediated opsonization, with or without the involvement of complement. However, some strains were reported to be resistant to opsonization, and a polysaccharide, possibly a capsular component, was identified as the mediator of resistance by several indirect observations (Arduino, Murray, & Rakita, 1994; Huebner, et al., 1999; Rakita, et al., 2000; Rakita, et al., 1999). For example, exposure of E. faecium strain DO (also known as TEX16) to sodium periodate, but not proteases or phospholipases, eliminated resistance to opsonization (Arduino, Murray, & Rakita, 1994). However, antibodies raised against this strain that targeted the carbohydrate fraction, also eliminated opsonic resistance, which restored the neutrophil killing of this strain (Rakita, et al., 2000).

Identification of an E. faecalis capsule was achieved by the purification of a carbohydrate that was localized to the surface of the bacterium when using specific antibodies. A genetic locus, cps for capsule polysaccharide, which encodes the genes necessary to generate this carbohydrate, was also identified (Hancock & Gilmore, 2002). A strain with an isogenic mutation in one of the genes (cpsI) was more susceptible to opsonophagocytotic killing by neutrophils and was compromised in its ability to persist within the lymph nodes of a mouse, which shows that the capsule contributes to virulence in strain OG1RF. Moreover, when capsule-specific antibodies were included in the phagocytic assay, the capsule-producing strains of E. faecalis were more effectively targeted for killing, which suggests that such antibodies could have therapeutic indications (Hancock & Gilmore, 2002). Further work (Thurlow R. L., Thomas, Fleming, & Hancock, 2009) showed that a capsule was protective against C3 complement, opsonophagocytic killing by macrophages. C3 was shown to deposit equally well on encapsulated versus non-encapsulated cells by Western blot analysis, but the capsule appeared to mask C3 epitopes on encapsulated whole cells, as there was less C3-antibody binding, as determined by flow cytometry (Thurlow R. L., Thomas, Fleming, & Hancock, 2009). Additionally, a capsule was shown to mask epitopes associated with LTA, which is a common target of anti-enterococcal antibody production (see below) (Thurlow, Thomas, & Hancock, 2009). A significant decrease in TNFα by the macrophages was associated with encapsulated strains, likely due to the decrease in LTA-exposed epitopes (Thurlow R. L., Thomas, Fleming, & Hancock, 2009). Further work on the structure of the capsular material revealed it to be a diheteroglycan, and antibodies raised against this material protected mice from bacteremia caused by encapsulated strains (Theilacker, et al., 2011).

Lipoteichoic acid

Various typing schemes for distinguishing strains of differing properties have been developed, based on differences in cell surface structures. These typing methods have been confounded by changes to the taxonomic classification of enterococci (Sharpe, 1964), and by variation in non-polysaccharide epitopes (Maekawa, Yoshioka, & Kumamoto, 1992). Hufnagel et al. (Hufnagel, et al., 2004) proposed a new typing scheme in 2004 that classified a majority of the tested E. faecalis strains into one of four types: CPS-A, B, C, or D. Strains of serotypes A and B only contained the cpsAB genes in their capsule locus, while serotypes C and D strains possess an additional eight to nine genes. Further work defined CPS-C serotype strains as containing the cpsF gene, which encodes an enzyme that carries out a glucosylation only found in the CPS-C capsule (Thurlow, Thomas, & Hancock, 2009). It was believed that serotypes A and B possessed a capsular polysaccharide of a different chemical nature than serotypes C and D, based on a purification of capsular material from a serotype A strain (Wang, et al., 1999). However, further investigation revealed that the material was actually LTA that had been inadvertently modified during the purification procedure (Theilacker, et al., 2006). Antiserum to serotypes A and B contain antibodies against LTA, which is accessible due to a lack of a capsule in these strains (Huebner, et al., 1999; Theilacker, et al., 2006; Theilacker, et al., 2011; Thurlow, Thomas, & Hancock, 2009). E. faecalis LTA is an important cell-wall component that influences the way in which the immune system recognizes infection. Antibodies raised specifically against LTA resulted in opsonic killing (Huebner, et al., 1999; Theilacker, et al., 2011), and are protective in a mouse model of enterococcal infection (Huebner, Quaas, Krueger, Goldmann, & Pier, 2000). All sequenced strains of E. faecium contain cpsAB homologues, but not the other capsule genes, which suggests that its ability to make this type of a capsule is rare or non-existent.

Common cell wall polysaccharide: Epa, enterococcal polysaccharide antigen

The existence of a cell wall polysaccharide that is common to all strains of E. faecalis was first detected by a study that screened for common antigens produced during E. faecalis human infections. DNA fragments from E. faecalis strain OG1RF were expressed in E. coli, and the production of antigenic material was detected by using serum from infected humans (Xu, Jiang, Murray, & Weinstock, 1997). One immunopositive clone produced proteinase K-resistant antigenic material, and subsequent study confirmed that the material was a carbohydrate (Xu, Jiang, Murray, & Weinstock, 1997; Xu, Murray, & Weinstock, 1998). Sequencing of the clone revealed a locus in which many of the genes encoded proteins with similarity to bacterial polysaccharide biosynthesis enzymes (Xu, Murray, & Weinstock, 1998). A later characterization defined the genes involved in generating the Epa polysaccharide as epaA–epaR, which corresponded to EF2198–EF2177 in the V583 genome (Teng, Singh, Bourgogne, Zeng, & Murray, 2009). Overall, by both predicted gene function and by compositional analysis of the antigenic material, the epa locus appears to be involved in generating a rhamnose-containing polysaccharide (Teng, Singh, Bourgogne, Zeng, & Murray, 2009; Xu, Murray, & Weinstock, 1998). The polysaccharide is hypothesized to be present in all strains of E. faecalis, as well as relatively invariant, based on sequence analysis of 12 strains (Teng, Jacques-Palaz, Weinstock, & Murray, 2002). A similar epa locus is found in all sequenced strains of E. faecium, except that three genes are missing (epaI, epaJ, epaK). Epa is postulated to reside deep in the cell wall due to difficulty in detecting it on the surface with antibodies, at least in vitro (Hancock & Gilmore, 2002; Xu, Murray, & Weinstock, 1998; Xu, Singh, Murray, & Weinstock, 2000).

Loss of the Epa polysaccharide resulted in attenuated virulence phenotypes in a variety of models and assays, which suggests that this polysaccharide contributes to the pathogenic properties of E. faecalis. Insertion mutants in epaB and epaE resulted in attenuated killing in a mouse peritonitis model with a two- to three-fold increase in the LD50 (Xu, Singh, Murray, & Weinstock, 2000). These two mutants were also more susceptible to neutrophil-mediated phagocytosis and killing, which suggests that this cell wall polysaccharide contributes to E. faecalis evasion of the immune system (Teng, Jacques-Palaz, Weinstock, & Murray, 2002). The polysaccharide may also contribute to colonization and invasion of tissue, as the epa mutants were additionally deficient in biofilm formation on a polystyrene surface (Mohamed, Huang, Nallapareddy, Teng, & Murray, 2004) and translocation across a polarized monolayer of colon epithelial cells (Zeng, Teng, Weinstock, & Murray, 2004). Consistent with a role in colonization and biofilm formation, the epaB mutant was also defective in a mouse model of ascending UTI (Singh, Lewis, & Murray, 2009).

Metabolic Characteristics

Bacterial adaptation to growth in a host as a pathogen inevitably results in metabolic changes, as the organism acclimates to niche-specific nutritional availabilities and arms itself to thrive in the face of the host immune response. Transcriptional microarray analysis of E. faecalis OG1RF harvested after eight hours of incubation in a rabbit subdermal abscess infection model revealed the differential regulation of nearly 300 genes, nearly 90% of which were down-regulated (58). Analysis of the down-regulated genes, which included those that coded for ribosomal proteins, aminoacyl tRNA synthetases, DNA replication enzymes, RNA polymerase subunits, ATP synthase machinery subunits, cell division-related proteins, and acyl carrier proteins, suggested activation of the stringent response among bacteria during infection in the subdermal abscess environment—although the stressor that triggered such a reaction remains unidentified. The stringent response has been shown to be necessary for E. faecalis infection in the C. elegans killing model (Abranches, et al., 2009). In E. faecalis, (p)ppGpp, the alarmone whose accumulation activates the stringent response during nutrient deprivation or environmental stress is synthesized by the enzymes RelA and RelQ. RelQ maintains (p)ppGpp levels during homeostatic growth, whereas RelA produces (p)ppGpp during stress conditions (Abranches, et al., 2009). Analysis of OG1RF ΔrelA, ΔrelQ, and ΔrelAΔrelQ in-frame deletion mutants in the subdermal abscess model demonstrated that both relA and relQ affect survival in this environment (Frank, Lemos, Schlievert, & Dunny, 2012). The ΔrelA strain, which displays higher basal levels of (p)ppGpp, exhibited increased survival during the early hours of infection, which suggests a protective role for the alarmone in the initial stages of infection. In contrast, ΔrelAΔrelQ and ΔrelQ showed reduced abilities to persist in the abscesses for several days, which suggests that production or maintenance of basal (p)ppGpp is a key factor for persistence. Further analysis of the same mutants in rabbit endocarditis showed that ΔrelA is severely attenuated, but ΔrelAΔrelQ , which has no (p)ppGpp, is not attenuated (Frank, Lemos, Schlievert, & Dunny, 2012). This phenotype indicates (p)ppGpp levels affect virulence and that the ability to hydrolyze (p)ppGpp is indispensible for E. faecalis to infect heart valves.

The ability to produce extracellular superoxide is a common trait among E. faecalis isolates, particularly those that cause bacteremia, and is infrequently found in E. faecium isolates (Huycke, Joyce, & Wack, 1996). It has been hypothesized that extracellular superoxide produced by intestinal commensal E. faecalis cells may be a potential source of chromosomal instability (CIN) in colonocytes, which may result in colorectal cancer (Huycke, Abrams, & Moore, 2002). An experiment that measured increased DNA damage in the colon cells of rats colonized with wild-type E. faecalis, as compared to those colonized with an isogenic superoxide non-producing strain, provided data in support of this hypothesis (Huycke, Abrams, & Moore, 2002). After further study, a detailed mechanism has emerged that states that E. faecalis extracellular superoxide production activates the release of DNA-damaging agents from activated macrophages, which results in CIN and cell cycle arrest in nearby epithelial cells (Wang, et al., 2008; Wang & Huycke, 2007; Wang, et al., 2012). In effect, this line of research, which is described in more detail elsewhere in this volume, suggests that superoxide-producing E. faecalis commensals induce bystander effects, which may ultimately contribute to the pathogenesis of colorectal carcinogenesis (Wang, et al., 2012).

Another metabolite associated with virulence in E. faecalis, at least in the C. elegans model, is ethanolamine (Maadani, Fox, Mylonakis, & Garsin, 2007). Ethanolamine can serve as a source of both carbon and nitrogen for those microbes that are equipped to catabolize it. Ethanolamine is present in large quantities in the intestine, and the ability to utilize this compound has been associated with intestinal pathogens, such as Salmonella species and Listeria monocytogenes (Garsin D. A., 2010). Though loss of one of the E. faecalis genes necessary to metabolize ethanolamine caused an attenuated phenotype in the worm model (Maadani, Fox, Mylonakis, & Garsin, 2007), it remains unclear whether the ability to metabolize ethanolamine affects E. faecalis intestinal commensalism or pathogenesis in mammals.

Other Factors Affecting Virulence of E. faecalis

Despite extensive research on enterococcal pathogenicity, few virulence factors present in all clinical isolates have been identified. This fact illustrates that infection is the result of complex interactions between factors derived from the host, as well as from the microbe. Enterococcal virulence depends on strain-variable combinations of factors belonging to the pan and core genomes that result in infection when expressed together. This includes factors that confer fitness inside the infected host.

Pathogens that invade a host are exposed to numerous stresses, and stress resistance has been linked to virulence. An interesting protein implicated in stress resistance identified in recent years is the general stress protein Gls24, of unknown function. This 20 kDa protein was initially identified to be strongly induced in stationary phase cells, and also in cultures exposed to bile salts or the heavy metal cadmium in E. faecalis strain JH2-2 (Giard, Rince, Capiaux, Auffray, & Hartke, 2000). The gls24 gene forms an operon with glsB, which encodes a small polypeptide of unknown function. This genetic organization is highly conserved among sequenced E. faecalis strains. The gls24 and glsB genes can also be expressed as a large mRNA that includes 4 other cistrons (orf1–orf4) upstream of gls24 (Giard, Rince, Capiaux, Auffray, & Hartke, 2000; Teng, Nannini, & Murray, 2005). The ORF that precedes gls24 (orf4) is a close paralog of gls24. Of note, E. faecalis strain V583 harbors a second, albeit incomplete orf1orf4 operon (orf1 is missing in this copy) situated in the pathogenicity island. The induction of gls24 expression in `stationary phase is due to promoter P2 situated immediately upstream of gls24, whereas bile salts treatment causes induction of the remote promoter P1, which is located in front of orf4. Stationary phase cultures of gls24 mutants constructed in two different strains are more sensitive to bile salts exposure than the parental strains (Giard, Rince, Capiaux, Auffray, & Hartke, 2000; Teng, Nannini, & Murray, 2005). Of greater importance, the gls24 disruption mutant (but not a glsB mutant) constructed in strain OG1RF was highly attenuated in a mouse peritonitis virulence model (Teng, Nannini, & Murray, 2005) and a rat endocarditis model (Nannini, Teng, Singh, & Murray, 2005). In this last study, a separate experiment also showed also that when administrated as a mixture with the parental strain, the gls24 mutant, but not the glsB mutant, was out-populated at the end of the experiment in vegetations, organs, and blood, despite being inoculated in greater numbers.

Phagocytes form the front line of defense used to fight invading pathogens. These cells have specific enzymes that generate antimicrobial reactive oxygen species (ROS). As a consequence, antioxidant defense activities of pathogens play an important role in colonization and persistence at the site of infection. E. faecalis is well equipped to survive oxidative stress in vitro, and is also highly resistant to intracellular killing by phagocytic cells (La Carbona, et al., 2007; Verneuil, et al., 2006). Part of this resistance is due to a manganese superoxide dismutase (Mn-SOD) (Verneuil, et al., 2006), a heme-dependent catalase (Frankenberg, Brugna, & Hederstedt, 2002) and three NADH-dependent peroxidases (alkylhydroperoxide reductase, thiol peroxidase, and NADH-peroxidase) (La Carbona, et al., 2007). Mutants with an inactivated sodA gene (that encodes the Mn-SOD), and with inactivated genes that encode the peroxidases (ahpCF, tpx, and npr), have been constructed and tested for attenuation in infection (La Carbona, et al., 2007; Verneuil, et al., 2006). These studies showed that the sodA and tpx mutants were highly compromised in survival inside mouse peritoneal macrophages. In addition, reduced survival inside microglial cells was recently shown for the sodA mutant (Peppoloni, et al., 2011), and attenuated virulence in a mouse peritonitis model was found for the Tpx-deficient strain (La Carbona, et al., 2007).

When antioxidant defense systems fail to protect the cell against ROS, damage repair pathways are activated. Methionine (Met) side chains of proteins are particularly vulnerable to oxidation, as they form methionine sulfoxide (MetSO). Methionine sulfoxide reductases A and B are antioxidant-repair enzymes that reduce methionine sulfoxides back to methionine (Zhao, et al., 2010). Deficiency of either Msr-enzyme in E. faecalis reduced survival inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide (but not in naïve phagocytes), and reduced virulence in a systemic and urinary tract infection (UTI) model (Zhao, et al., 2010).

Several proteins implicated in host-pathogen interactions are characterized by a leucine-rich repeat (LRR) domain. The best characterized of these LRR proteins are IlnA and IlnB from Listeria monocytogenes, which trigger internalization into cells (Hamon, Bierne, & Cossart, 2006). Two internalin-like proteins were identified in E. faecalis V583 (EF2250 and EF2686), both of which are characterized by an additional C-terminal WxL domain involved in peptidoglycan binding (Brinster, et al., 2007). The polypeptide encoded by ef2686, named ElrA for Enterococcus leucine-rich protein A, was recently shown to be important for E. faecalis virulence, since a disruption mutant was significantly attenuated in a mouse peritonitis model, demonstrated reduced survival in macrophages, and displayed a decreased interleukin-6 response in vivo (Brinster, et al., 2007).

Several transcriptional regulators that are likely to control determinants important for virulence or fitness have also been identified in E. faecalis. The disruption of hypR, which encodes a regulator that shows some sequence homology to the main oxidative stress regulator OxyR of E. coli, reduced resistance to oxidative stress, survival in in vivo infected peritoneal macrophages, and virulence in a mouse peritonitis model (Verneuil N. , et al., 2005; Verneuil, et al., 2004). Additionally, inactivation of a homologue of PerR, the most important transcriptional regulator of the oxidative stress response of Bacillus subtilis, decreased virulence of the mutant in the peritonitis model (Verneuil N. , et al., 2005). Using transposon mutagenesis, a new gene locus implicated in biofilm formation has been identified (Hufnagel, Koch, Creti, Baldassarri, & Huebner, 2004). This locus is composed of four genes that are likely expressed as an operon, designated bop (biofilm on plastic surfaces). The transposon was inserted into the second gene of this operon, and the corresponding mutant showed reduced biofilm production and decreased persistence in a mouse bacteremia model. The last gene of the operon encodes a transcriptional regulator belonging to the LacI/GalR family, which was less expressed in the transposon mutant and was responsible for the observed phenotypes (Hufnagel, Koch, Creti, Baldassarri, & Huebner, 2004). An AraC-type transcriptional regulator, designated PerA and encoded by a gene located within the pathogenicity island, was also implicated in biofilm formation and virulence. A corresponding mutant constructed in the clinical isolate E99 produced more biofilm, but was less pathogenic in a mouse peritoneal infection model and was attenuated for survival within macrophages in vitro (Coburn, Baghdayan, Dolan, & Shankar, 2008). The enterococcal regulator of survival (Ers), a member of the Crp/Fnr family of transcriptional regulators, is a close homologue of PrfA, which is the main regulator of virulence genes in Listeria monocytogenes. Survival of a mutant with a disrupted ers gene within peritoneal macrophages was highly reduced and the strain was also less virulent in a mouse peritonitis model (Giard, et al., 2006; Riboulet-Bisson, et al., 2008). In contrast, the mutation of slyA, which encodes a transcriptional regulator of the MarR/SlyA family, led to increased virulence of the mutant strain in the Galleria mellonella model, better survival inside in vivo infected mouse macrophages, and longer persistence in mouse kidneys and livers (Michaux, et al., 2011). A strain with a deleted sigV gene encoding an extracytoplasmic function (ECF) sigma factor was shown to have decreased persistence in both the kidneys and liver, and in a UTI model, showed reduced colonization of the kidneys and bladders (Le Jeune, et al., 2010).

Factors Contributing to Virulence in E. faecium

Over the past three decades, there has been a gradual shift towards a higher proportion of enterococcal infections being caused by E. faecium (from ~5% to more than 35%) in the USA (94) and in many European hospitals (Top, Willems, van der Velden, Asbroek, & Bonten, 2008; Werner, et al., 2008). Besides resistance to antibiotics, including ampicillin and vancomycin, it appears that other properties have enhanced the hospital adaptation and infectivity of E. faecium, such as traits that contribute to colonization of the intestinal tract, translocation through the intestinal wall, and/or attachment to internal organs and other tissue sites. A significant challenge to studying E. faecium pathogenesis has been difficulty in engineering specific mutations, which stems in part from the scarcity of selection markers for use in antibiotic-resistant clinical strains, as well as their poor transformability. Some of these obstacles have been overcome by the introduction of conjugative, temperature-sensitive plasmids and positive counterselection systems (Kristich, Chandler, & Dunny, 2007; Nallapareddy, Singh, & Murray, 2006). Thus far, only three genes/loci, acm (Nallapareddy, Singh, & Murray, 2008), the ebpfm (Sillanpää J. , et al., 2010) operon, and esp (Heikens, Bonten, & Willems, 2007; Heikens, et al., 2011), have been shown to play a role in E. faecium virulence when tested in animal models.

Acm

Adherence studies with E. faecium have shown that, like E. faecalis, many isolates can adhere to collagen, but typically do so after growth in a routine laboratory medium (BHI) (Nallapareddy, Weinstock, & Murray, 2003), in contrast to the conditional adherence exhibited by most E. faecalis isolates (Xiao, Höök, & Weinstock, 1998). A highly significant association was found in collagen adherence of E. faecium isolates of clinical origin, versus community fecal or animal origin, which suggests that such adherence to collagen may contribute to the ability of E. faecium to persist, colonize, and cause infection in the hospital environment (Nallapareddy S. R., Singh, Okhuysen, & Murray, 2008; Nallapareddy, Weinstock, & Murray, 2003). Analysis of an early draft version of the first E. faecium genome (strain TX16, also known as DO) (Fox, 2000) identified acm (adhesin of collagen from E. faecium), a gene that encodes an MSCRAMM family protein with higher similarity to the S. aureus MSCRAMM Cna than to Ace of E. faecalis (Nallapareddy, Weinstock, & Murray, 2003). Recombinant Acm was subsequently shown to bind to collagen type I and, to a lesser degree, type IV, but not to laminin, fibrinogen, or fibronectin (Nallapareddy, Weinstock, & Murray, 2003). The introduction of a functional acm gene into two collagen adherence-negative natural E. faecium acm mutants, on a low-copy-number shuttle vector, led to a collagen adherence phenotype for both strains (Nallapareddy, Weinstock, & Murray, 2003). Acm was later confirmed to mediate collagen adherence of E. faecium using an acm deletion mutant, which nearly eliminated the collagen binding of strain TX82 (Nallapareddy, Singh, & Murray, 2006). Although the acm gene is carried by almost all E. faecium isolates, it occurs as a functional gene almost exclusively in multi-drug resistant E. faecium isolates of clinical origin, while it is present as an inactive pseudogene, often interrupted by a transposon, in approximately one quarter of isolates from non-clinical sources (Nallapareddy, Singh, & Murray, 2006; Nallapareddy S. R., Singh, Okhuysen, & Murray, 2008). Moreover, the level of collagen adherence correlates strongly with the amount of Acm produced on the cell surface of diverse E. faecium isolates, which supports the role of Acm as the primary collagen adhesin of E. faecium, at least in vitro.

A recent study by Nallapareddy et al. (Nallapareddy, Singh, & Murray, 2008) reported that the acm deletion mutant of strain TX82 was highly attenuated in experimental endocarditis using a rat model, both in early adherence/colonization of the heart valve, and in established vegetations, while no differences in mortality were seen in a mouse peritonitis model. Further examination of 17 endocarditis isolates revealed that, although 5 of them did not produce detectable surface Acm nor adhere to collagen, all had an intact acm sequence that was highly identical to those of Acm-producing isolates. Not surprisingly, mRNA levels of acm were reduced in some of these Acm non-producers (e.g., TX16) under standard in vitro growth conditions, explaining the lack of surface Acm and collagen adherence (Nallapareddy, Singh, & Murray, 2008). However, flow cytometry analysis of extracts processed directly from rat vegetations infected with TX16, detected Acm on ~40% of TX16 cells, which demonstrates the active production of Acm during infection. This finding is consistent with the presence of antibodies against Acm in the serum of the endocarditis patient infected with TX16 (Nallapareddy, Singh, & Murray, 2008). A larger survey of patient sera found anti-Acm antibodies in all sera from E. faecium endocarditis patients, and in most sera from patients with other E. faecium infections, despite the lack of Acm expression or collagen adherence by some of the infecting strains, which suggests that Acm expression in vivo is common even by those acm+ clinical isolates that do not express it in vitro (Nallapareddy S. R., Singh, Okhuysen, & Murray, 2008). Anti-Acm antibodies purified from an endocarditis patient serum were able to significantly inhibit the adherence of E. faecium to collagen (Nallapareddy, Singh, & Murray, 2008), similar to rabbit antibodies raised against the collagen-binding subdomains of Acm (Nallapareddy S. R., Sillanpää, Ganesh, Hook, & Murray, 2007). These studies support the concept of Acm as a potential target for developing antibody-based strategies for immunoprophylaxis, or in combination with antibiotics to treat E. faecium infections. A further possibility could be active or passive immunization with of a mixture of two or more E. faecium surface adhesins, perhaps in combination with the ubiquitous and highly conserved E. faecalis Ace and/or Ebp pili, for broader coverage of both major enterococcal pathogens.

Ebpfm pili

Recent analyses of the first available draft E. faecium genome (endocarditis strain TX16/DO) identified 15 genes (including acm) that encode LPXTG-motif, cell-wall anchored proteins with MSCRAMM-like characteristics (see Enterococcal cell wall components and structures). These include a cluster of three pilin-encoding genes, initially named ebpAfm, ebpBfm, and ebpCfm. Although the gene organization of the ebpfm locus, including a downstream sortase gene, is nearly identical to the ebp-bps locus of E. faecalis, these encoded proteins show relatively large amino acid sequence divergence between these two species, with similarities ranging from 66% (EbpBfm vs. EbpB) to 85% (EbpCfm vs. EbpC) (Nallapareddy S. R., et al., 2011). Also, transcriptional organization of the two ebp operons were shown to be different, with separate ebpABCfm and bpsfm transcripts produced by E. faecium compared to a four-gene ebpABC-bps transcript, plus an additional bps transcript, produced by E. faecalis (Nallapareddy S. R., et al., 2006; Sillanpää J. , et al., 2008). Using an ebpABCfm deletion mutant, this operon was recently shown to be important for the ability of E. faecium TX82 to cause infection in an ascending UTI model, as well as to form biofilm (Sillanpää J. , et al., 2010), which indicates functional similarity between the Ebp type pili of E. faecium and E. faecalis. However, it is currently unclear whether the Ebpfm pili have a similar role in endocarditis and adherence to host collagen, fibrinogen, and platelets, as shown by their counterpart in E. faecalis. Although E. faecium TX16 has been shown/predicted to produce three additional pili, as well as individual MSCRAMMs (see Enterococcal cell wall components and structures)—with several of these genes reported to be enriched in isolates of the hospital-associated clade versus those in the community clade—direct evidence for their involvement in virulence is lacking.

Esp

Similar to E. faecalis, E. faecium clinical isolates are enriched in an esp homolog that shares >90% amino acid identity (Leavis, et al., 2004) and is encoded by a pathogenicity island (Leavis, et al., 2004; Willems, et al., 2001). The esp gene encodes a very large LPXTG-motif cell-wall anchored protein that represents one of the few identities within the pathogenicity islands of these two species. Studies with an esp deletion mutant showed that as for E. faecalis, esp of E. faecium contributes to biofilm formation (Heikens, Bonten, & Willems, 2007), virulence in UTI in a mouse model (Leendertse, et al., 2009), and virulence in endocarditis (Heikens, et al., 2011), similar to previous reports on esp of E. faecalis. No contribution for esp was detected in mouse peritonitis (Leendertse, et al., 2009), intestinal colonization (Heikens, et al., 2009), or adherence to the intestinal epithelial cell line Caco-2 (Heikens, et al., 2009).

Other factors

Increased virulence of E. faecium transconjugants that harbor conjugative megaplasmids has been reported in mouse peritonitis (Arias, Panesso, Singh, Rice, & Murray, 2009) and colonization models (Rice, et al., 2009). In both models, these plasmids were found to contain the hyaluronidase hyl gene, which was previously associated with a higher prevalence in clinical isolates, as compared to community isolates. However, the subsequent deletion of hyl caused no effect on peritonitis, which suggests that this model bypasses a step where it contributes, or that other uncharacterized virulence factors are encoded by the hyl-containing plasmid, and this gene is a passive marker in virulent strains (Panesso, et al., 2011).

Two homologs of the E. faecalis general stress protein, Gls24 (Giard, Rince, Capiaux, Auffray, & Hartke, 2000; Nannini, Teng, Singh, & Murray, 2005; Teng, Nannini, & Murray, 2005), have recently been characterized in E. faecium (Choudhury, Singh, Sillanpää, Nallapareddy, & Murray, 2011). The genes that encode these proteins, gls33 and gls20 (named according to the calculated molecular weights of their encoded proteins), are both followed by homologs of E. faecalis glsB (designated as glsB and glsB1 in E. faecium), and are part of larger operons that are only partially similar to the gls operon of E. faecalis. Comparisons among sequenced Efm genomes revealed clade-specific differences between the gls33 and gls20 operons, corresponding to ~7% nucleotide sequence difference between hospital-associated (HA) and community-associated (CA) gls33 operons, and ~3.5% difference between HA and CA gls20 operons (Choudhury, Singh, Sillanpää, Nallapareddy, & Murray, 2011; Galloway-Peña, Rice, & Murray, 2011). This genetic divergence between the HA and CA lineages is in agreement with that recently reported for most of the MSCRAMM- and pilus-encoding genes of E. faecium, as well as the clade-specific differences seen at the core genome level (see Enterococcal Genomics). A double deletion mutant that lacked both gls33-glsB and gls20-glsB1, but neither single deletion mutant, was shown to be highly attenuated in a mouse peritonitis model, while mutants that lack either gls33-glsB or gls20-glsB1 or both were all more sensitive to bile salts than the wild-type parent strain (Choudhury, Singh, Sillanpää, Nallapareddy, & Murray, 2011). Similar attenuation has previously been demonstrated with a gls24 mutant of E. faecalis in experimental peritonitis (Teng, Nannini, & Murray, 2005) and endocarditis (Nannini, Teng, Singh, & Murray, 2005), in addition to a decrease in bile salts tolerance (Giard, Rince, Capiaux, Auffray, & Hartke, 2000; Nannini, Teng, Singh, & Murray, 2005), which demonstrates that the gls loci of both species are important for virulence, and suggests their involvement in adaptation to the intestinal environment. Finally, protection studies using anti-Gls24 serum against experimental peritonitis by E. faecalis suggest Gls proteins as useful targets for immunotherapy.

Conclusions

In the strictest definition, “virulence factor” is defined as a substance that is necessary for causing disease in the host, but not necessary for survival in other contexts. Toxins, like the one produced by Bacillus anthracis and other toxin-producing pathogens, mostly fit this criterion. While a significant number of genetic determinants contribute to the ability of a given enterococcal strain to cause infection, these determinants are not necessarily found in every clinical isolate, which highlights the point that enterococcal infection is multifactorial and involves contributions by the microbe, as well as the host. Many enterococcal factors that contribute to fitness in the host also contribute to the overall fitness of the bacterium in other ecologies, including its normal habitat, the GI tract. The virulence of enterococci is more complex than the simple presence of some main players, and appears to be dependent on strain-variable combinations of factors that lead to improved infection and colonization when expressed together in the right background. Virtually all studies on enterococci as pathogens have focused on the genetic factors that contribute to their pathogenic potential, with fewer studies focusing on the role of the host or on factors that contribute to the commensal lifestyle of enterococci. Promoting such noninfectious behavior could be a novel strategy for potentially preventing and treating infections. Understanding how enterococci contribute to the human microbiome as well as infection would help to illuminate exactly where they occur on the commensal-pathogen continuum.

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Bookshelf ID: NBK190426PMID: 24649512

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