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Mol Microbiol. Author manuscript; available in PMC 2010 Dec 1.
Published in final edited form as:
Mol Microbiol. 2009 Dec; 74(6): 1459–1470.
PMCID: PMC2814410
NIHMSID: NIHMS165668

The Francisella tularensis Pathogenicity Island Encodes a Secretion System that is required for Phagosome Escape and Virulence

Summary

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella Pathogenicity Island (FPI), a cluster of genes that we show here shares homology with Type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation, and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

Introduction

F. tularensis is a highly infectious bacterium that causes tularemia in humans, a disease that has a high mortality rate when acquired through the pulmonary route. Very little is known about the pathogenic mechanisms employed by this organism to cause disease and avoid host immunity, and no vaccine is currently available for human use in the U. S. F. tularensis is able to survive and replicate within host macrophages, and this ability is essential for virulence. After entry into macrophages, F. tularensis escapes from the phagosomal compartment and replicates within the macrophage cytosol (Golovliov et al., 2003). Cytosolic bacteria induce the caspase-1-dependent inflammasome, resulting in apoptosis (Mariathasan et al., 2005), and the bacteria are re-engulfed in autophagosomes (Checroun et al., 2006), yet despite these attempts at host defense the bacteria are capable of high levels of intracellular replication and rapid dissemination within the host. F. tularensis appears to induce broad immunosuppression within the host (Bosio et al., 2007) as proinflammatory cytokine expression is notably repressed during early stages of infection (Telepnev et al., 2005), and infected cells are unable to respond to TLR-dependent secondary stimuli (Hajjar et al., 2006). These data strongly suggest F. tularensis expresses effector(s) that modulate the host immune response.

Intracellular growth and virulence of F. tularensis are dependent upon the Francisella Pathogenicity Island (FPI), a cluster of 17 genes that is duplicated in the high virulence (for humans) subsp. tularensis and subsp. holarctica strains, and found in single copy in the low virulence (for humans) subsp. novicida (Nano et al., 2004). The FPIs share >97% nucleotide identity across the different subspecies. One of the genes within the FPI encodes IglC, one of the most upregulated proteins during intramacrophage growth (Golovliov et al., 1997). IglC is required for phagosomal escape and evasion of phagosome-lysosome fusion (Santic et al., 2005, Lindgren et al., 2004), yet the exact function of IglC remains unknown. Additional FPI genes iglA, iglB, iglD, pdpA, and FTT1353 have also been shown to be required for intramacrophage growth and virulence (Nano et al., 2004, de Bruin et al., 2007, Santic et al., 2007, Brotcke et al., 2006). Transcription of the FPI genes is under the positive control of the global virulence regulator MglA (Lauriano et al., 2004).

The FPI proteins IglA and IglB share homology with proteins encoded within secretion gene clusters in other bacteria (de Bruin et al., 2007). Recently, gene clusters in Vibrio cholerae and Pseudomonas aeruginosa containing iglA and iglB homologues have been characterized as novel Type VI (T6SS) secretion systems (Mougous et al., 2006, Pukatzki et al., 2006). In these T6SS clusters, icmF, clpV, and dotU genes are required for the secretion of Hcp and VgrG. ClpV is a member of the Hsp100/Clp ring-forming ATPases (Schlieker et al., 2005), and ClpV has been demonstrated to hydrolyze ATP and aggregate into discrete foci within P. aeruginosa under conditions conducive to Type VI secretion (Mougous et al., 2006). A serine-threonine kinase, PpkA, and serine-threonine phosphatase, PppA, encoded within the P. aeruginosa T6SS cluster, have antagonistic activities that stimulate and inhibit, respectively, Hcp secretion by regulating phosphorylation of another T6SS protein, Fha (Mougous et al., 2007). The function of the secreted factor Hcp is not yet clear, although its structure suggests it forms a channel. The V. cholerae secreted VgrG-1 protein is translocated into macrophages from phagocytosed bacteria, where it cross-links actin (Pukatzki et al., 2007, Ma et al., 2009). The IglA and IglB homologues VipA and VipB in V. cholerae have recently been shown to form tubular structures that are remodeled by ClpV into small complexes, and this is proposed to be an essential step in T6S (Bonemann et al., 2009).

No direct role for the P. aeruginosa or V. cholerae T6SS in human disease has yet been identified, although long-term cystic fibrosis patients infected with P. aeruginosa have antibodies against Hcp, and the V. cholerae T6SS is required for virulence in Dictyostelium discoideum (Mougous et al., 2006, Pukatzki et al., 2006). However, T6SS in Burkholderia mallei has been shown to secrete Hcp and to be required for virulence in a hamster model of infection (Schell et al., 2007), and a T6SS in Edwardsiella tarda secretes VgrG, Hcp, and a third protein EvpP, and this secretion system is essential for virulence in fish (Zheng & Leung, 2007). Also, an Hcp homolog in Aeromonas hydrophila is secreted into host cells via a T6SS, and this contributes to virulence in the septicemic mouse model.(Suarez et al., 2008).

In the current study, we analyzed the FPI gene cluster in F. tularensis, and found homology with the T6SS clusters in V. cholerae and P. aeruginosa. We demonstrate that the F. tularensis VgrG homologue is secreted into host macrophages, and that this FPI protein is required for phagosomal escape, intramacrophage growth, and virulence. Interestingly, VgrG secretion does not require other FPI genes; rather, VgrG and other FPI genes contribute to the secretion of another FPI protein, IglI, which is required for phagosomal escape, as well as the induction of IL1-β expression. Our studies demonstrate that the FPI encodes a secretion system responsible for modulating the host cell to allow F. tularensis to replicate and cause disease.

Results

The Francisella Pathogenicity Island proteins share homology with T6SS proteins

The FPI is a cluster of 17 genes that are required for intramacrophage growth and virulence of F. tularensis (Nano et al., 2004). The iglA and iglB genes share homology with genes found in secretion loci of a number of bacteria, including the T6SS clusters of V. cholerae (VCA0107 and VCA0108) and P. aeruginosa (PA0083 and PA0084), as noted previously (de Bruin et al., 2007). Bioinformatic analysis of the FPI genes identified additional homology with other T6SS genes, including icmF (pdpB; FTT1345), vgrG (FTT1347), clpV (FTT1348), dotU (FTT1351), and hcp (FTT1355) (Fig. 1A; alignments in Fig. S1), suggesting that the FPI may represent a T6SS-like gene cluster. The other genes within this cluster that have either not been previously named or that share no obvious homology to T6SS genes, we propose to rename intramacrophage growth locus (igl) genes, because we have obtained evidence that each of these genes is required for intramacrophage growth (see below and J. Liu and K. Klose unpublished). We have utilized F. tularensis subsp. novicida (Ftn) in the characterization of FPI components, due to the relative ease of genetic manipulation in this subspecies, in part because of the presence of only a single FPI. The Ftn FPI shares high homology (>97% nucleotide identity) to the duplicated FPIs found in F. tularensis subsp. tularensis and subsp. holarctica (Ftt and Fth), indicating that the functional attributes of the encoded proteins are likely identical between the subspecies.

Figure 1
Comparison of F. tularensis FPI with T6SS gene clusters in Pseudomonas aeruginosa and Vibrio cholerae

No FPI homologues were found to the T6SS kinase and phosphatase genes ppkA and pppA. Additionally, the F. tularensis ClpV homologue FTT1348 lacks the characteristic Walker A and B boxes found in the P. aeruginosa and V. cholerae ClpV proteins (Schlieker et al., 2005), indicating that the F. tularensis ClpV lacks the ATPase activity shown to be essential for T6SS in P. aeruginosa (Mougous et al., 2006). Also, the Walker A box present in the IcmF homologues is missing from F. tularensis PdpB (Zheng & Leung, 2007). Finally, the F. tularensis VgrG homologue is much smaller (17.5 kD) than the V. cholerae VgrG-1 (128.8 kD), VgrG-2 (77.2 kD), and VgrG-3 (113 kD) proteins, sharing limited homology to the region encompassing aa 415–643 in these proteins (Fig. 1B and S1). This homology spans portions of the bacteriophage tail spike-like gp27 and gp5 domains, which Pukatzki et al. proposed functions like a “cell-puncturing device” for delivery of effector proteins (Pukatzki et al., 2007). Notably, the F. tularensis VgrG lacks the C-terminal extensions found in the V. cholerae VgrG-1 and VgrG-3 proteins, one of which (VgrG-1) contains an actin cross-linking domain.

F. tularensis VgrG is secreted into macrophages

One of the current hallmarks of T6SS is that the Hcp and/or VgrG proteins are secreted into the supernatants, as seen in P. aeruginosa, V. cholerae, E. tarda, and A. hydrophila, and secretion is clpV-, dotU-, and icmF-dependent (Mougous et al., 2006, Pukatzki et al., 2006, Zheng & Leung, 2007, Suarez et al., 2008). Moreover, V. cholerae cells translocate VgrG-1 into the J774 macrophage cytosol, which induces actin cross-linking because VgrG-1 contains an actin cross-linking domain (Pukatzki et al., 2007, Ma et al., 2009). To determine whether Hcp and/or VgrG are translocated into the macrophage cytosol of F. tularensis-infected J774 cells, we constructed plasmids that express protein fusions of Hcp and VgrG to the B. pertussis adenylate cyclase toxin (Hcp-CyaA and VgrG-CyaA). Because CyaA requires calmodulin for adenylate cyclase activity, elevated cAMP levels only occur in macrophages infected with F. tularensis expressing the CyaA fusion proteins if they are translocated into the macrophage cytosol (Sory & Cornelis, 1994). As evidence of this principle, Ftn expressing PepO (a secreted protease; (Hager et al., 2006)) fused to CyaA causes elevated cAMP levels within infected J774 cells (Fig. 2A). In contrast, Ftn expressing beta-lactamase from which the 17 aa N-terminal signal sequence has been removed (ΔNBlaB) fused to CyaA has no effect on J774 cAMP levels, as expected since this protein is confined to the bacterial cytoplasm. Significantly elevated cAMP levels were measured in J774 cells infected with Ftn expressing VgrG-CyaA, indicating that VgrG is secreted into the macrophage cytosol. However, cells infected with Ftn expressing Hcp-CyaA showed no significant increase in cAMP over that seen with Ftn expressing ΔNBlaB-CyaA, indicating that Hcp-CyaA is not secreted into the macrophage. Western immunoblot analysis indicated that all CyaA fusion proteins were being expressed at similar levels within Ftn (Fig. S2A).

Figure 2
Secretion of F. tularensis FPI proteins

The FPI contains genes with homology to T6SS components, as discussed above. To determine whether any of these genes are required for secretion of VgrG-CyaA into macrophages, we first constructed a Ftn strain with a complete deletion of the entire FPI [ΔFPI; Δ (pdpA-pdpD); Materials & Methods], and then infected J774 cells with the ΔFPI strain expressing VgrG-CyaA. Elevated cAMP levels could be detected in these infected cells as well, and the levels were similar to that seen in J774 cells infected with the wildtype Ftn strain expressing VgrG-CyaA (Fig. 2A). These results demonstrate that the putative T6SS genes in the FPI are not required for secretion of VgrG into macrophages.

VgrG is secreted into culture supernatants

The VgrG homologues in V. cholerae and P. aeruginosa are secreted into culture supernatants (Mougous et al., 2006, Pukatzki et al., 2006). To determine whether secreted VgrG could be detected in Ft culture supernatants, we examined filtered culture supernatants of the wildtype U112 strain and the Ftn vgrG mutant KKF102 (see below) expressing N-terminally FLAG-tagged VgrG (FLAG-VgrG) by anti-FLAG Western immunoblot (Fig 2B). FLAG-VgrG could be detected in the culture supernatants of the wildtype and vgrG mutant strains, indicating that VgrG is secreted during in vitro growth, and that no N-terminal processing is required for secretion of Ft VgrG. These are both attributes of T6SS characterized in other bacterial species. The Ftn strain lacking the entire FPI (ΔFPI) also secreted FLAG-VgrG into culture supernatants at levels similar to that of the vgrG strain. These in vitro results are consistent with the results obtained in vivo with VgrG-CyaA in macrophages (Fig. 2A) that indicated the FPI genes are not required for VgrG secretion. Ftn expressing ΔNBlaB with a C-terminal FLAG tag (ΔNBlaB-FLAG) had no detectable ΔNBlaB in the culture supernatant (Fig 2B), demonstrating a lack of cell lysis under the growth conditions being tested. Western immunoblot of the whole cell lysates revealed that all strains expressed relatively high levels of FLAG-tagged proteins.

VgrG is required for F. tularensis phagosomal escape, intramacrophage growth, and virulence

To determine the role of VgrG in F. tularensis pathogenesis, a vgrG Ftn strain was constructed (Materials & Methods) and evaluated for virulence parameters. The vgrG strain KKF102 was defective for growth within the J774 macrophage cell line (Fig. 3A), exhibiting low levels of intracellular bacteria 24 and 48h post infection, in comparison with the wildtype strain U112, which replicates to high intracellular levels during this time period. The intracellular growth defect of the vgrG strain was overcome by expression of FLAG-VgrG in trans. The intracellular growth of the complemented mutant was not identical to the wildtype strain, likely due to the altered stoichiometry caused by the multicopy plasmid and/or the constitutive promoter used to express vgrG in trans. These results demonstrate that VgrG is required for intramacrophage growth, and that FLAG-tagged VgrG maintains its normal function.

Figure 3
Intramacrophage growth and Phagosome Escape of F. tularensis FPI mutant strains

To examine further the intracellular defects of the vgrG mutant, we subjected infected mouse bone marrow-derived macrophages (BMM) to a phagosomal integrity assay based on differential fluorescent labeling of cytoplasmic bacteria and bacteria within intact phagosomes ((Checroun et al., 2006); Fig. 3B, representative images in Fig. 3D). Most wildtype Ftn bacteria were cytoplasmic by 4 h post infection (>90%). In contrast, the vgrG mutant remained largely within intact phagosomes at 4 h post infection, similar to an iglC and a ΔFPI mutant, and consistent with previous reports on iglC mutants (Santic et al., 2005, Lindgren et al., 2004). The phagosome escape defect of the vgrG mutant was overcome by expression of FLAG-VgrG in trans within this strain, resulting in the majority of bacteria having escaped the phagosome by 4 h post infection.

The vgrG mutant was highly attenuated for virulence, exhibiting an LD50 of >6 × 106 CFU when administered intranasally to Balb/C mice (7/7 mice survived at this inoculum with no outward signs of infection). In contrast the wildtype Ftn strain has an LD50 of <10 CFU when administered similarly, as has been reported previously (Santic et al., 2005, Lindgren et al., 2004). Collectively these results demonstrate that VgrG is critical for Ft phagosome escape, intramacrophage growth, and virulence.

Detection of secreted VgrG within infected macrophages

To visualize VgrG within infected macrophages, the vgrG Ftn strain expressing FLAG-VgrG was used to infect J774 macrophages, and VgrG was visualized using a Cy3 (red)-labelled anti-FLAG monoclonal antibody at 40 minutes post infection (Fig. 4). VgrG could be visualized within the macrophage cytosol around and clearly distinct from the bacteria, which were labeled with anti-Fn LPS (green) (Fig. 4B; see confocal reconstruction movies in supplemental data). In contrast, ΔNBlaB-FLAG expressed by Ftn within J774 macrophages colocalized with the bacteria, with no secreted protein detectable, as anticipated (Fig. 4A). FLAG-VgrG expressed by the ΔFPI mutant within J774 cells could still be visualized around and distinct from the bacteria (Fig. 4C), indicating VgrG secretion from the ΔFPI mutant strain. The FLAG-VgrG appears in closer proximity to the ΔFPI mutant, which remains within the phagosomal compartment, than to the (complemented) vgrG mutant, which is able to escape the phagosome (Figs. 3B and 3D). Surface mapping of the Ftn and VgrG fluorescent signals (IMARIS; Bitplane Scientific Solutions) was consistent with VgrG secretion from Ftn within infected macrophages (see Fig. S3).

Figure 4
Detection of VgrG and IglI within infected macrophages by immunofluorescence

VgrG and the FPI are required for secretion of IglI in macrophages

We hypothesized that, given their homology with T6SS proteins, VgrG and FPI genes might be required for the secretion of other proteins encoded within the FPI, and suspected that some FPI genes with no obvious homology to T6SS genes may represent secretion substrates. The iglI gene (FTT1352) encodes a hypothetical protein of 44.6 kD with no obvious homology to T6SS genes. A Ftn mutant strain lacking iglI was defective for intramacrophage growth within J774 cells, and complementation of this strain with FLAG-tagged IglI restored intramacrophage growth (Fig. 3A).

To determine whether the intracellular growth defect of the iglI mutant was due to failure of this mutant to escape the phagosome, we subjected infected mouse BMM to the phagosomal integrity assay ((Checroun et al., 2006); Fig. 3C, representative images in Fig. 3D). Unlike the wildtype strain, the iglI mutant remained largely within intact phagosomes at 4 h post infection, similar to the vgrG mutant. The phagosome escape defect of the iglI mutant was overcome by expression of FLAG-IglI in trans within this strain, resulting in the majority of bacteria having escaped the phagosome by 4 h post infection, demonstrating that IglI is critical for phagosomal escape. The iglI mutant exhibited an LD50 of >4.6 × 106 CFU when administered intranasally into Balb/C mice (7/7 mice survived at this inoculum with no outward sign of infection), which is at least 105-fold greater than the LD50 of the wildtype strain, indicating that IglI is required for Ftn virulence.

Infection of J774 macrophage cells with the Ftn wildtype strain expressing a IglI-CyaA fusion led to a significant increase in cAMP, indicating secretion of IglI-CyaA into the macrophage cytosol (Fig. 2A). Importantly, infection of J774 macrophage cells with the vgrG or ΔFPI mutant strains expressing IglI-CyaA did not cause a significant increase in cAMP compared to cells infected with Ftn alone, or expressing ΔNBlaB-CyaA, demonstrating that vgrG and other factors within the FPI are required for secretion of IglI into macrophages. IcmF homologues have been shown to be important for T6S in several bacteria (Mougous et al., 2006, Pukatzki et al., 2006), so we also measured cAMP levels in macrophages infected with a strain lacking the IcmF homologue PdpB (ΔpdpB) expressing IglI-Cya. This pdpB/icmF strain also failed to cause elevated cAMP levels in infected macrophages, similar to the ΔFPI strain, consistent with the T6S homologue IcmF being required for IglI secretion into macrophages.

Examination of culture supernatants from the WT strain expressing FLAG-IglI revealed that FLAG-IglI is also secreted in vitro (Figs. 2B and S2C). FLAG-IglI secretion into culture supernatants was clearly reduced in the vgrG, pdpB/icmF, and ΔFPI mutant strains, although low levels of FLAG-IglI could still be detected within the supernatants of these strains. These results demonstrate that VgrG, PdpB/IcmF, and the FPI contribute to secretion of IglI, in macrophages as well as in vitro.

To determine if some specific cellular compartment is targeted by IglI within infected cells, we constructed a vector that expresses a fusion of IglI to dsRed Fluorescent Protein (dsRed-IglI), transfected HeLa cells with this construct, and performed fluorescence microscopy on the transfected cells, looking for co-localization with markers for cellular components. There was no evidence for targeting of early endosomes, lysosomes, endoplasmic reticulum, or actin cytoskeleton by dsRed-IglI expressed within eukaryotic cells (Fig. S3).

VgrG and IglI are required for induction of IL1-βexpression by infected macrophages

F. tularensis phagosomal escape into the macrophage cytosol is critical for the induction of IL-1β release, a result of inflammasome activation (Mariathasan et al., 2005, Gavrilin et al., 2006). Because VgrG and IglI are required for phagosomal escape, we determined whether VgrG and IglI are also required for Il1-β expression by infected macrophages. Mouse-derived BMM were infected with Ftn strains, and IL1-β release was measured at 5, 10, and 24 h post-infection, as described (Materials & Methods). Macrophages infected with the wildtype Ftn strain induced IL1-β release (Fig. 5), and IL1-β release was abrogated in macrophages infected with an FPI mutant or an mglA mutant (which regulates the FPI) as has been shown previously (Weiss et al., 2007, Gavrilin et al., 2006). Macrophages infected with the vgrG and iglI mutant strains also failed to release IL1-β, consistent with the role of VgrG and IglI in phagosome escape. Complementation of the vgrG strain with FLAG-VgrG, which restored phagosomal escape (Fig. 3), also restored IL1-β release by infected macrophages (Fig. 5). Likewise, complementation of the iglI strain with FLAG-IglI, which restored phagosomal escape (Fig. 3), also restored IL1-β release by infected macrophages (Fig. 5). Release of IL1-β by macrophages infected with the complemented vgrG and iglI strains was lower relative to IL1-β release by wildtype Ftn-infected macrophages, which correlates with the delayed phagosomal escape exhibited by these strains (Fig. 3). Our results are consistent with the VgrG (and FPI)-dependent secretion of IglI facilitating F. tularensis phagosomal escape, leading to cytosolic replication, inflammasome induction, and virulence.

Figure 5
IL-1β signalling in macrophages infected with F. tularensis FPI mutants

Discussion

The ability of F. tularensis to escape the phagosome and replicate within the macrophage cytosol is essential for its virulence, and yet the molecular mechanism(s) underlying this ability are still not clear. Several genes within the FPI gene cluster are critical for phagosome escape and intramacrophage growth (Lindgren et al., 2004, Mariathasan et al., 2005, Santic et al., 2005), and the duplication of the FPI within the most virulent strains indicates the importance of these genes for F. tularensis pathogenesis. We show here that a number of FPI proteins share homology with recently described T6SS components of V. cholerae, P. aeruginosa, and E. carta, which suggested that these proteins may also represent secretion components of F. tularensis. Moreover, we demonstrate that the FPI proteins VgrG and IglI, which are required for phagosome escape, intramacrophage growth, inflammasome activation, and virulence in mice, are secreted into macrophages. Several reports have suggested that the FPI encodes a secretion apparatus (Nano et al., 2004, Broms et al., 2009, Ludu et al., 2008), but this is the first demonstration of FPI-dependent secretion of virulence proteins into macrophages.

F. tularensis VgrG is a small 17.5 kDal protein that shares limited homology with a portion of the VgrG proteins from V. cholerae (aa 414–640 of Vc VgrG; Fig. S1). This region of the V. cholerae VgrG proteins spans portions of the “gp27-like” and “gp5-like” junction, with homology to phage T4 tail-spike proteins. Studies in V. cholerae have revealed that VgrG-1 contains an actin cross-linking domain at its C-terminal end (not present in Ft VgrG; Fig. 1B), and that V. cholerae within the macrophage phagosome translocates VgrG-1 into the cytosol to cause actin cross-linking (Pukatzki et al., 2007, Ma et al., 2009). The authors speculate that VgrG acts as a “cell-puncturing device” for delivery of effector proteins through the membranes of target host cells (Leiman et al., 2009).

We have demonstrated that Ft VgrG is secreted into culture supernatants, like V. cholerae VgrG proteins, and moreover we have shown that Ft VgrG is secreted into the macrophage by intracellular bacteria. However, secretion of Ft VgrG, either into culture supernatants or into the macrophage, did not require any of the other genes within the FPI, which includes homologues to IcmF, ClpV, DotU, and Hcp, all of which are required for secretion of VgrG into culture supernatants of V. cholerae, P. aeruginosa, and E. tarda (Mougous et al., 2006, Pukatzki et al., 2006, Zheng & Leung, 2007, Suarez et al., 2008). This suggests that the proteins encoded within the FPI may not function in an identical fashion to those within the T6SS of V. cholerae, P. aeruginosa, and E. tarda, which is perhaps not surprising given the limited amount of homology of F. tularensis VgrG with the V. cholerae VgrG proteins (Fig. 1B). However, in these other systems, VgrG contributes to the T6SS-dependent secretion of other factors, and that function appears to be conserved in Ft VgrG. We demonstrate here that Ft VgrG contributes to secretion of IglI into macrophages. Collectively, these results suggest VgrG is a component of the secretion “apparatus”.

Only low levels of Ft VgrG and IglI could be detected within culture supernatants Detectable in vitro secretion of VgrG in P. aeruginosa required the introduction of chromosomal mutations (retS), or only occurred in specific V. cholerae strains (Vc strain V52) (Mougous et al., 2006, Pukatzki et al., 2006), suggesting that Type VI-dependent secretion into culture supernatants naturally occurs at low levels in these bacteria, and is likely upregulated only under specific inducing conditions. Secretion of the FPI proteins is likely also upregulated upon entry into macrophages, since expression of the FPI protein IglC has been shown to be upregulated in this environment (Golovliov et al., 1997). We suspect that the VgrG-, IcmF(PdpB)-, and FPI-dependent IglI secretion we detected within macrophages reflects the (high-level) induced secretion necessary for intramacrophage growth, while the (low-level) VgrG- and FPI-(semi)-independent secretion of IglI in culture supernatants reflects basal level secretion.

IglI encodes a 383 aa protein with no obvious homology to proteins found in other organisms. IglI is required for phagosome escape, and subsequent cytosolic replication and induction of inflammasome-dependent IL1-β release. Thus the primary function of IglI is likely destabilization/degradation of the phagosome membrane. IglI expressed within eukaryotic cells failed to detectably co-localize with early endosomes, lysosomes, or endoplasmic reticulum (Fig. S3), suggesting that the protein does not specifically target membranes in the absence of the other FPI proteins. We have also shown that tags at both the N- and C-termini do not prevent IglI secretion, indicating a lack of proteolytic processing, similar to T6SS substrates characterized previously (Mougous et al., 2006, Pukatzki et al., 2006, Schell et al., 2007, Zheng & Leung, 2007, Suarez et al., 2008). Neither VgrG nor IglI is secreted in E. coli (Fig. S2B), indicating a requirement for expression in F. tularensis for secretion. We suspect that IglI must be secreted through the FPI-dependent secretion pathway in order to correctly function at its cellular target. Because IglI secretion within macrophages requires VgrG, IcmF/PdpB, and other FPI factors, and the iglI mutant is defective for intramacrophage growth and mouse virulence, its function within the macrophage is likely to yield insight into fundamental pathogenic mechanisms of F. tularensis.

Our results demonstrate that VgrG and other FPI factors facilitate secretion within macrophages, and share some similarity with T6SS in other bacteria. However, some of the notable differences of the FPI with T6SS gene clusters in other bacteria mentioned above may derive from the fundamentally different pathogenic strategies of these bacteria: Ft is an intracellular pathogen, whereas V. cholerae and P. aeruginosa are extracellular pathogens. E. tarda and A. hydrophila also have dominant extracellular phases in infected fish and/or humans, though whether these organisms also have a significant intracellular phase is still unclear (Han et al., 2006). Thus F. tularensis may have evolved these T6SS homologues to facilitate secretion of effector proteins in a related, but not identical manner, adapted to its location within the host phagosomal compartment. The T6SS was originally identified by the ability of V. cholerae to kill the protist D. discoideum (Pukatzki et al., 2006), and we have previously shown that FPI genes are required for intraamoebal growth as well as intramacrophage growth (Lauriano et al., 2004), thus we speculate that T6SS and related systems such as that encoded by the FPI represent an ancestral defense mechanism against protists that has been adapted for pathogenesis of higher eukaryotes.

Materials and Methods

Ethics Statement

All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the UTSA IACUC.

Strains and Media

All Ftn strains are isogenic with strain U112. A complete strain list can be found in Table S1. Construction of strains KKF34 (ΔmglA::ermC), KKF24 (ΔiglC::ermC), KKF255 (ΔpilF::ermC), and KKF215 (ΔpepO::ermC) has been described previously (Lauriano et al., 2003, Zogaj et al., 2008). Construction of Ftn mutants KKF101 (ΔpdpB::ermC), KKF102 (ΔvgrG::ermC) and KKF108 (ΔiglI::ermC) was performed as previously described (Liu et al., 2007), with primers listed in Table S3. Construction of the Ftn ΔFPI strain KKF219 was achieved by introducing FLP-recombinase recognition target (FRT) sites into both ends of the FPI, then expressing FLP-recombinase within this strain to mediate excision of the intervening DNA. This resulted in the deletion of 17 FPI genes [Δ (pdpA-pdpD)]; the details of this strain construction can be found in supplemental data. Strains were grown on TSAP broth/agar (Liu et al., 2007), or Chamberlain’s media (Chamberlain, 1965) supplemented with10 μg/ml Tetracycline (Sigma) or 100 μg/ml erythromycin, as appropriate. Plasmids were electroporated into Ftn as previously described (Zogaj et al., 2008).

Plasmid Construction

Plasmids used in this study were derived from Francisella plasmids pKK214 (Kuoppa et al., 2001). A complete plasmid list can be found in Table S2. In all plasmids the FTN1451 promoter (Gallagher et al., 2007) was used to express vgrG-cyaA (pKEK1012), hcp-cyaA (pKEK1016), iglI-cyaA (pKEK1051), pepO-cyaA (pKEK939), ΔNblaB-cyaA (pKEK1072), FLAG-vgrG (pKEK1157), FLAG-iglI (pKEK1175), and FLAG-ΔNblaB (pKEK1191). Specific details on the construction of these plasmids can be found in supplemental data.

Intracellular Survival and Adenylate Cyclase Assay

Intracellular growth in the J774 macrophage cell line was assayed as previously described (Lauriano et al., 2003). Determination of adenylate cyclase activity in J774 cells infected with Ftn expressing CyaA-tagged proteins was performed as previously described (Sory & Cornelis, 1994). Briefly, J774 macrophages were seeded at 105 cells/ml and incubated at 37° C with 5% CO2 overnight. Macrophages were infected at an MOI of 10:1 for 1h. Media was aspirated and fresh media was added with 50 μg/ml Gentamicin for 1h. Macrophages were washed with fresh media and allowed to incubate for a total of 4 hours. Quantification of cAMP levels was determined using the cAMP Biotrak Enzymeimmunoassay (EIA) System (Amersham Biosciences) according to manufacturers’ instructions.

Protein Detection in Culture Supernatants

Ftn strains were grown in TSAP to an OD600 ~ 1.0. Cultures were centrifuged (7270 × g) 30 min at 4° C. Supernatant fractions were filtered (0.2μM) and precipitated with Trichloracetic Acid (10% final), then incubated overnight at 4° C and centrifuged (7270 × g) for 2 h. The pellet was washed with 50 ml of acetone, centrifuged (7270 × g) 15 min, acetone was removed and the pellet was allowed to dry overnight. Pellets were resuspended in 2X SDS-PAGE buffer and separated by 12% SDS-PAGE, then subjected to Western immunoblot utilizing mouse monoclonal anti-FLAG antibody (Sigma); detection was performed with the ECL detection reagent (GE Healthcare).

Measurement of IL1-β expression

Bone marrow-derived macrophages (BMM) were prepared from 4-week old C57BL/6 mice as previously described (Bosio & Elkins, 2001). BMM were seeded into 24 well culture plates at a density of 2×106 cells per well in 0.5 ml of DMEM plus 10% fetal bovine serum (D-10) 2 hrs before infecting with 2×108 Ftn strains. The plates were incubated for 2 hrs, washed with DMEM, and then incubated with D-10 containing gentamicin (10 μg/ml) for 1 hr to kill any remaining extracellular bacteria. The media was then replaced with D-10 and supernatants were collected at 5, 10, and 24 hrs, and assayed for IL-1β content using OptEIA kit (BD Biosciences, San Diego, CA) according to manufacturer’s recommendation.

Mouse Virulence Assays

Groups of seven female BALB/c mice (Harlan Sprague) were inoculated intranasally with Ftn strains in 20 μl PBS (Lauriano et al., 2004). Actual bacterial numbers delivered were determined by plate count from inocula and an approximate LD50 was determined from surviving mice. Mice were monitored for 30 days after infection.

Immunofluorescence Microscopy

J774 cells were infected with Ftn at an MOI of 10:1 until desired time point. Samples were washed 3x with PBS and fixed using 3% paraformaldehyde, then permeabilized using 0.5% Triton X-100. Samples were incubated with anti-Ftn LPS (Immuno-Precise Antibodies, Limited) antibodies (1:1000) conjugated to Alexa Fluor 488 (molecular probes) and anti-FLAG-CY3 (Sigma) antibodies (1:5000) and DAPI for 1 h. Samples were washed 3x with PBS and mounted using FluorSave (Calbiochem), then visualized using a Zeiss LSM 510 Meta confocal microscope and data was analyzed with IMARIS software.

Phagosomal Integrity Assay

Assay was performed as previously described (Checroun et al., 2006) with the following modification. Ftn was detected with mouse anti-Ftn monoclonal antibody 8.2 (Immuno-Precise Antibodies, Limited).

Supplementary Material

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Acknowledgments

We thank Rebecca Henry and Nisha Hariharan for construction of plasmids, Coleen Witt for assistance with microscopy, and Yufeng Wang for assistance with bioinformatics.

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