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Infect Immun. Feb 1999; 67(2): 740–744.
PMCID: PMC96381

Impact of the High-Affinity Proline Permease Gene (putP) on the Virulence of Staphylococcus aureus in Experimental Endocarditis

Editor: V. A. Fischetti

Abstract

Staphylococcus aureus causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567–572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive S. aureus infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (putP) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (104 to 106 CFU) in terms of achievable intravegetation densities (P, <0.05). The negative impact of putP inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original putP mutant and the parental strain as well as by challenge with a putP mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of S. aureus.

The pathogenesis of infective endocarditis (IE) involves a complex interaction between the organism and a variety of host vascular surfaces, cells, and serum proteins (2, 4, 13, 15, 22, 23, 31, 40). Thus, for induction of IE, the microbe must first colonize damaged endothelial surfaces on cardiac valves by binding to platelets (22, 23, 40, 44) and diverse matrix proteins deposited at such endothelial sites (e.g., fibrinogen, fibrin, and thrombospondin [17, 21, 32]). The organism must then persist and proliferate at these initial sites of endothelial colonization; this latter phase of in vivo survival requires the microbe to both acquire necessary nutrients for ongoing growth and metabolism (16) and resist local host defenses mounted at these damaged endothelial sites (e.g., elaboration of antimicrobial peptides from platelets (11–13).

Establishing the role of putative virulence factors and genes in experimental invasive infections, such as IE, has proven to be a daunting task. Most such work has relied on comparing parental strains with isogenic mutants in which the gene(s) of interest has been inactivated by a variety of genetic techniques. For example, using Staphylococcus aureus as a prototypical pathogen in experimental IE, we and others have shown that the following genetic loci appear to contribute to the overall in vivo virulence of this pathogen in IE: sar, agr, hla (alpha-toxin structural gene), clf (clumping factor gene encoding cell surface fibrinogen-binding protein), and fnbAB (fibronectin-binding protein genes) (3, 79, 17, 32). However, the relative hierarchical roles of these individual genetic loci in S. aureus virulence have been difficult to establish.

Recently, several research groups developed a “negative selection” genetic strategy to simultaneously assess the relative impact of a large number of genetic loci in S. aureus on the capacity of the organism to survive in specific anatomic niches by use of diverse animal models (10, 19, 30, 42). These investigators used signature tag transposon mutagenesis (STM) with transposons tagged with unique oligonucleotides. This strategy enables the contemporaneous analyses of a large cadre of individual mutants within large heterogenous pools introduced into a specific animal model. One can then delineate the capacity of isogenic S. aureus clones within such mutant pools to survive and persist at diverse anatomic sites of interest. STM mutants which are viable in vitro in the infecting pool but cannot persist in vivo (i.e., in vivo negative selection) would therefore have transposon inactivation of an essential gene for in vivo staphylococcal survival.

Using this STM strategy, Coulter et al. (10) found that, among more than 1,500 oligonucleotide signature tag (OST) mutants evaluated in three murine models and one rabbit model of invasive S. aureus infections, only ~2% of these OST mutants were attenuated in vivo. The largest gene class identified by this study as impacting the in vivo survival of S. aureus encodes peptide and amino acid transporters (e.g., putP, encoding a high-affinity proline permease uptake system [42, 46, 49]). The aim of the current investigation was to directly compare the virulence of the parental S. aureus strain from the above-described OST mutant pool studies with a representative array of individual OST mutants identified as attenuated in vivo in multiple animal model systems. We used the rabbit IE model as the relevant and discriminatory system with which to carry out these comparisons of isogenic S. aureus OST strains.

MATERIALS AND METHODS

Strains. Table Table11 lists the strains and plasmids used in the current study. S. aureus RN4220 is a derivative of strain 8325-4 that can be transformed or transduced with foreign DNA (33). The virulent, hemolytic strain RN6390 (33) was used for final genetic transfers to construct the putP mutant, 16F-157 putP::Tn917, and other OST mutants (10, 42). Strain S6C (26) (provided by S. Projan, Pearl River, N.Y.) was used as a recipient strain for the transduction of the putP mutation into a different staphylococcal genetic background (S6C-3 putP::Tn917). S. aureus OST mutant strains 7F-857 (purR::Tn917), 16F-310 (oppC::Tn917), and 7F-1032 (epiA::Tn917) have been described elsewhere (10, 42). The phenotypic defects exhibited by the above OST mutants include proline uptake (16F-157 putP::Tn917 and S6C-3 putP::Tn917), oligopeptide transport (16F-310 oppC::Tn917), epidermin synthesis (7F-1032 epiA::Tn917), and deregulation of purine biosynthesis (7F-857 purR::Tn917).

TABLE 1
Bacterial strains and plasmid used

MAX efficiency Escherichia coli DH10B cells (Gibco/BRL, Gaithersburg, Md.) were used for marker rescue experiments of the flanking S. aureus DNA. Shuttle vector pMOD-1, used for delivery of the signature tag Tn917 elements into the S. aureus chromosome, has been described elsewhere (42).

S. aureus cells were grown in brain heart infusion (BHI; Gibco/BRL) medium, whereas E. coli cells were grown in Luria-Bertani medium (Gibco/BRL). The media were supplemented with antibiotics (Sigma Chemical Co., St. Louis, Mo.) at the following concentrations (in micrograms per milliliter): ampicillin, 100, tetracycline, 12.5, lincomycin, 5; chloramphenicol, 5; and erythromycin, 1 for OST mutants and 5 for transductants.

Construction and screening of OST mutants.

As previously noted (10, 42), a library of 1,520 S. aureus Tn917 mutants, divided into 16 pools of 95 individual OST mutants, was constructed for in vivo screening (25, 36). In vivo growth of the mutant pools was evaluated with three murine models of infection (abscess, burn wound, and bacteremia [10, 47]) by comparing their viability with that of pools of mutants grown in vitro (42). Four mutants that showed diminished in vivo growth in one or more of the animal models were further analyzed and are included in the current study (7F-857, 7F-1032, 16F-157, and 16F-310).

Marker rescue and sequencing of flanking genomic DNA.

Genomic DNA was prepared from each mutant strain to determine the site of Tn917 insertion within the staphylococcal chromosome as previously described (42). Briefly, digested genomic DNA was recircularized by ligation and was transformed into E. coli DH10B competent cells. Transformants containing the ampicillin resistance determinant within the integrated transposon were selected and grown to recover plasmid DNA. Plasmid DNAs were sequenced with an ABI 377 automated sequencer (Perkin-Elmer, Foster City, Calif.) by use of standard dideoxy termination chemistry (39) and a primer directed to the 5′-terminal repeat of Tn917. Additional primers directed to flanking genomic DNA were used to extend the sequence analysis of the transposon mutants. Sequencing results were analyzed with Sequencer software (Gene Code Corp., Ann Arbor, Mich.) and used to query public DNA and protein databases (1) by BLASTX and TBLASTN homology searches. In addition, putP genomic analysis of flanking sequences was done by comparison against the S. aureus genome (The Institute for Genomic Research, database; www.tigr.org).

Animal model of IE.

The rabbit model of experimental IE used in these studies has been described elsewhere (34). Briefly, anesthetized rabbits underwent transcarotid-transaortic valvular catheterization with an indwelling polyethylene catheter to induce sterile aortic valve vegetations. IE was produced by the intravenous (i.v.) injection of each of the S. aureus strains listed in Table Table11 at 24 h postcatheterization. To examine the relative capacities of these strains to induce IE, separate groups of catheterized rabbits were individually challenged i.v. with a range of inocula of each staphylococcal strain (104 to 106 CFU) that encompassed the 95% infective doses (ID95) of most S. aureus strains in this model (79). To generate adequate statistical power, at least seven catheterized rabbits were challenged with each staphylococcal strain at all challenge inocula. In a parallel study, mutants which demonstrated reduced virulence compared to the parental strain in the IE model were further evaluated in a competition study. In this strategy, 10 rabbits with aortic catheters were challenged at 24 h postcatheterization with 107 CFU of both the parental and mutant strains simultaneously in separate ear veins. This inoculum represents an ID100 challenge for both the parental and all mutant strains, as determined in pilot studies in our laboratory. Such simultaneous challenge strategies enabled a direct comparison of the survival capacities of the parent versus the mutants in a single anatomic niche (12).

Assessment of IE.

Rabbits were sacrificed 48 h after i.v. challenge with each staphylococcal strain. Rabbits were euthanatized with a rapid i.v. injection of sodium pentobarbital (Abbott Laboratories, North Chicago, Ill.). At sacrifice, proper catheter position was verified, and cardiac vegetations from individual animals were removed, weighed, pooled, homogenized, and then quantitatively cultured in Trypticase soy agar (TSA) as previously described (79). Intravegetation staphylococcal densities were expressed as log10 CFU per gram; culture-negative vegetations were considered to contain ≤2 log10 CFU/g, based on average vegetation weights of ~0.01 g (79). IE was considered as having been induced in any animal with culture-positive vegetations, irrespective of final vegetation density. For the simultaneous (competition) challenge studies, vegetation homogenates were plated in parallel on plain TSBA and TSBA containing erythromycin (10 μg/ml). This protocol takes advantage of the erythromycin resistance determinant within the Tn917 transposon used in this study (10) and enables the specific quantification of parental versus mutant staphylococcal densities within the same vegetative lesion (12).

Statistical analyses.

Statistical comparisons were performed with regard to the mean staphylococcal densities within infected vegetations in animals challenged with parental S. aureus strains or various signature tag transposon mutants. Kruskal-Wallis analysis of variance was used, with correction for multiple comparisons via the Tukey post hoc modification. A P value of ≤0.05 was considered significant.

RESULTS

The parental S. aureus strain used in this study (RN6390) induced IE in 40, 70, and 100% of animals at challenges of 104, 105, and 106 CFU, respectively. There were no significant differences in the overall capacities of the various OST mutants and the parental strain to induce IE (data not shown). Moreover, there were no significant differences in the mean weights of the infected vegetations induced by the parental strain or any of the transposon mutants studied (data not shown). As noted in Table Table2,2, in the individual challenge studies, only putP OST mutant 16F-157 exhibited evidence of substantially reduced virulence compared to the parental strain in terms of the capacities to persist and proliferate within vegetative lesions. Thus, the achievable staphylococcal vegetation densities for the putP mutant were significantly lower than that for the parental strain at all three challenge inocula. To further verify this observation, animals with aortic catheters were simultaneously challenged with 107 CFU of both the parental strain and strain 16F-157 (representing more than the ID100 inoculum, to ensure 100% infection rates for both strains). The achievable vegetation densities for the OST mutant strain (6.41 ± 2.20 log10 CFU/g [mean ± standard deviation for 10 animals]) were again significantly lower (P, 0.05) that those for the parental strain (7.72 ± 0.70 log10 CFU/g for 10 animals).

TABLE 2
Vegetation densities in animals challenged with parental strain RN6390 and STM mutants of S. aureus

To provide further supportive evidence concerning the impact of the putP mutation on virulence in the IE model, we compared the achievable vegetation densities of two additional S. aureus strains, parental strain S6C and its isogenic variant, S6C-3, transduced with the putP mutation (individual challenge strategy). This latter mutant exhibits the same phenotype (i.e., decreased proline uptake) as the RN6390-derived transposon mutant 16F-157. As shown in Table Table3,3, parental strain S6C was substantially more virulent than parental strain RN6390, achieving significantly higher vegetation densities at all challenge inocula (P, <0.05). However, even in this strain, with a high level of intrinsic virulence in the IE model, the presence of the putP mutation still rendered the strain less virulent than the parental strain. The achievable vegetation densities for mutant strain S6C-3 were significantly lower than those for its parental strain, S6C, at two of the three challenge inocula (105 and 106 CFU).

TABLE 3
Achievable vegetation densities for a parental strain of S. aureus and a transduced isogenic variant carrying the putP mutation

The genomic organization of the putP locus was evaluated for possible polar transcription effects. Using the amino acid sequence of the putP gene to query the S. aureus TIGR database, an 8,517-bp sequence was found to contain the putP locus, as well as open reading frames (ORFs) both 5′ and 3′ of the locus in question. Upstream of putP are two ORFs that are transcribed in the opposite orientation. These ORFs have strong sequence homology (<e−164) to the Bacillus subtilis subunit A and B genes for Glu-tRNA Gln amidotransferase (locus accession no. AF008553). Immediately 3′ of the putP locus and also transcribed from the opposite coding strand are ORFs homologous to the B. subtilis gene yerH, encoding a DNA helicase (<e−70; locus accession no. Y15254), and a DNA ligase gene (<e−62; locus accession no. AJ011676) from Bacillus stearothermophilus. Given that all of the ORFs immediately flanking the putP gene are transcribed from the opposite coding strand, this genomic configuration reveals that polar transcription effects from a transposon insertion within putP are unlikely.

DISCUSSION

The staphylococci are the most common collective cause of both community-acquired and nosocomial bloodstream infections worldwide, accounting for ~40% of such events in most studies (27, 35, 43). The staphylococci cause a variety of serious localized infections (e.g., wound infections and skin and soft-tissue abscesses [27]) and bacteremic infections, especially in selected patient populations (persons with diabetes mellitus and intravenous drug addicts [27]) and in certain infectious disease syndromes (e.g., IE and hemodialysis vascular access site infections [27]). Importantly, a substantial portion of cases of severe staphylococcal infections are now being caused by strains which are resistant to the semisynthetic, antistaphylococcal penicillins (i.e., oxacillin-resistant strains [19, 28]), aminoglycosides, rifampin, and/or quinolone agents (19, 28, 38). Of particular concern is the recent documentation of significant and recalcitrant clinical infections due to oxacillin-resistant strains with reduced susceptibility to vancomycin in vitro in diverse geographic areas in the United States (5, 6) as well as in Japan (24). The rising incidence of invasive bacteremic S. aureus infections and the evolving problem of multiantibiotic resistance in S. aureus underscore the critical necessity for the development of newer antistaphylococcal therapies and strategies.

A recent popular venue for the development of novel antimicrobial approaches has been the identification of essential genes vital to the survival of the microbe of interest. The prevailing concept is that the delineation and characterization of such genetic loci will provide a template and a target for the rational design of novel antimicrobial agents. To accomplish this goal, the STM technique has proven to be very useful. In this strategy, pools of transposon mutants with unique oligonucleotides are injected into relevant animal models to define specific genetic mutations which render the mutants less able to survive in vivo (negative selection [20]). This strategy was successfully used recently by several groups to identify a number of candidate essential gene targets in S. aureus (10, 30, 42). For example, Lei et al. (30), using a murine peritonitis infection model, identified two broad categories of essential genes in S. aureus which had an impact on in vivo survival: genes involved in nutrient biosynthesis (e.g., tryptophan, lysine, and threonine synthesis) and genes involved in cell wall assembly (e.g., peptidoglycan cross-linking). Coulter et al. (10) and Schwan et al. (42) extended this approach. These investigators took the realistic perspective that a single animal model would have a low likelihood of being an accurate identifier of genetic loci which are universally important for S. aureus survival in vivo. By use of multiple animal infection models (bacteremia, wound, abscess, and IE), a consensus panel of mutated genetic loci which resulted in attenuated persistence of the organism in vivo in diverse anatomic niches and in distinct animal species was identified. The largest gene class delineated by screening of STM mutant pools in multiple animal models included genes encoding amino acid or oligopeptide transporters. Such transporter genes include the functionally diverse ATP-binding cassette (ABC) transporter protein genes.

S. aureus requires an organic source of nitrogen supplied by 5 to 12 essential amino acids, including proline (29). Thus, since most S. aureus strains are innately auxotrophic for a number of these essential amino acids (including proline), it is likely that transport mutants are not able to normally import selected amino acids or oligopeptides from which growth-essential amino acids are salvaged. Moreover, the transport of amino acids such as proline appears to be critical for the maintenance of normal osmoregulation in S. aureus (46). In this regard, the putP system of E. coli (in which the PutP transport protein shows substantial sequence homology to the S. aureus PutP protein) is a sodium-cotransport system (46). Thus, it has been suggested that the putP system in S. aureus may well represent the first gram-positive member of a sodium-solute symporter superfamily (37, 50).

The notion of a critical role for proline uptake in S. aureus survival in vivo was confirmed by Schwan et al. (42) in a detailed analysis of the putP OST transposon mutant which exhibited in vivo attenuation in multiple animal models. The putP gene encodes the high-affinity proline permease transporter system (46, 49). The putP mutation led to an ~33% decline in the capacity of the mutant strain to take up [3H]proline in vitro, compared to the capacity of the parental strain. Furthermore, the blockade of normal proline uptake in the parental strain by synthetic proline analogues attenuated the in vitro growth profiles of the organism. Moreover, direct assessment of the in vivo virulence of the putP mutant strain compared to that of the parental strain in an infected murine wound model, as well as in a murine abscess model, confirmed its attenuated virulence compared to that of the parental strain (42). The introduction of the transposon mutation in putP could have had polar effects on downstream genes that in turn caused the attenuation of virulence observed in these latter murine models. However, the ORFs flanking the monocistronic putP ORF on the S. aureus genome are coded for on the opposite DNA strand relative to putP. The fact that the putP ORF is divergent in orientation from the flanking ORFs indicates that there are no polar effects on these neighboring genes and that the transposon insertion in putP is responsible for the in vivo attenuation observed in our rabbit endocarditis model.

The results from the current study further support the concept that the putP mutation in S. aureus has a significant negative impact on the ability of this pathogen to survive in vivo. First, vegetation densities achieved by strains exhibiting the putP mutation in two distinct genetic backgrounds were significantly lower than those observed for each corresponding parental strain. Second, this reduction in virulence exhibited by the putP mutants was seen at multiple challenge inocula in the IE model. Finally, the attenuated virulence observed with the putP mutants was observed during either individual or simultaneous in vivo challenge with the parental strains. Durack and Beeson (16) previously showed that bacteria within the depths of experimental cardiac vegetations have a diminished capacity to import a variety of essential amino acids, including proline. It is thus quite likely that this model will magnify in vivo any defects in the capacity of S. aureus to import essential amino acids in vitro.

Other OST mutants used in the current study which were previously shown to be attenuated in multiple animal models (including the IE model) when included in OST mutant pools (10) failed to exhibit attenuation when administered individually in the IE model. This group of OST mutants had mutations in the epiA gene, involved in lantibiotic biosynthesis (41), the ABC transporter gene oppC (45), and the purine repressor gene purR (48). A previous OST mutant pool screening in the IE model (42) did not examine the overall vegetation densities for each of the mutants, and compensatory effects in a diverse mutant pool could have biased the population data as a whole. This pool effect could have been particularly important for the 7F-1032 mutant, which has a defect in the synthesis of epidermin, a small cationic peptide with antistaphylococcal activity (41). Because of this mutation in epiA and potential polar effects in its operon, this mutant would likely be susceptible to the staphylocidal effects of lantibiotics produced by the other OST mutants in the mutant pool. Thus, in a pooled population, this mutant would be markedly attenuated. However, if this strain were inoculated individually (as in the current IE model), there would be no lantibiotic secretion emanating from competing OST strains to potentially attenuate its intravegetation proliferation.

In summary, these data underscore the utility of the OST negative selection methodology for identifying candidate genetic loci with a substantial impact upon microbial survival in diverse anatomic niches and distinct physiologic microenvironments. These studies also emphasize the need to independently test all such candidate STM mutants identified by large-pool animal model screens as attenuated in separate and relevant animal models, as in the current investigation.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the National Institute of Allergy and Infectious Diseases to A.S.B. (RO1-AI39108).

We thank Cong Li and Iri Kupferwasser for excellent technical assistance in the animal model studies.

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