A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.     

SsaM and SpiC interact and regulate secretion of Salmonella pathogenicity island 2 type III secretion system effectors and translocators

The type III secretion system (TTSS) encoded by Salmonella Pathogenicity Island 2 (SPI-2) is required for systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. The SPI-2 TTSS is activated after internalization of bacteria by host cells, and translocates effector proteins into and across the vacuolar membrane, where they interfere with several host cell functions. Here, we investigated the function of SsaM, a small protein encoded within SPI-2. An ssaM deletion mutant had virulence and intracellular replication defects comparable to those of a SPI-2 TTSS null mutant. Although the ssaM mutant was able to secrete the effector protein SseJ in vitro, it failed to translocate SseJ into host cells, and to secrete the translocon proteins SseB, SseC and SseD in vitro. This phenotype is similar to that of a strain carrying a mutation in the SPI-2 gene spiC, whose product is reported to be an effector involved in trafficking of the Salmonella vacuole in macrophages. Both ssaM and spiC mutants were found to oversecrete the SPI-2 effector proteins SseJ and PipB in vitro. Fractionation assays and immunofluorescence microscopy were used to investigate the localization of SsaM and SpiC in macrophages. No evidence for translocation of these proteins was obtained. The similar phenotypes of the ssaM and spiC mutants suggested that they might be involved in the same function. Pull-down and co-immune precipitation experiments showed that SpiC and SsaM interact within the bacterial cell. We propose that a complex involving SsaM and SpiC distinguishes between translocators and effector proteins, and controls their ordered secretion through the SPI-2 TTSS.     

SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins

Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.     

The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking

The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome-lysosome fusion in vivo, and it is sufficient to inhibit endosome-endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST-SpiC pulled down Hook3 from murine macrophages, and anti-Hook3 antibodies precipitated SpiC from the cytosol of Salmonella-infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant-negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome-lysosome fusion.     

Host cell processes that influence the intracellular survival of Legionella pneumophila

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.     

Intracellular activities of Salmonella enterica in murine dendritic cells

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.     

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.     

Trafficking of the Salmonella vacuole in macrophages

Salmonella enterica is a facultative intracellular pathogen which can replicate in macrophages. Intracellular Salmonella exist in a membrane-bound compartment called the Salmonella -containing vacuole. Most studies on Salmonella trafficking in relation to the endocytic pathway have concluded that the majority of Salmonella -containing vacuoles do not interact extensively with late endosomes and lysosomes. Numerous bacterial genes have been identified which are required for survival and replication in macrophages. These include the spv operon, located on the large virulence plasmid, the phoP-phoQ regulon, and those connected with the Salmonella pathogenicity island 2 type III secretion system. The functions of some of these genes are beginning to be understood. In this review, I discuss their roles in relation to our broader understanding of Salmonella trafficking in macrophages.     

Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms-aroE intragenic region

Salmonella-induced aggregation of host endosomal compartments into tubules, termed lgp-tubules, requires sifA and ompR. Lgp-tubules result from Salmonella-directed alteration of the endocytic system and typify the unique intracellular locale where Salmonella replicate. A high-throughput method devised to screen 11 520 MudJ mutants for loss of lgp-tubule formation identified one auxotrophic and nine prototrophic mutants. Molecular characterization identified four new loci required to alter epithelial endocytic structure. Salmonella pathogenicity island 2 (SPI2) is the locus central to the phenotype. A subset of SPI2 effectors is essential: SpiC and SseFG are required, but not SseE. A subset of apparatus proteins is also implicated: SsaJ, L, M, V and P are required. SPI2 was implicated further, as SifA shows similarity with known SPI2 translocation targets, and OmpR regulates SPI2. Another locus lies within the smf-aroE intragenic region. Lgp-tubule formation also involves a locus on the virulence plasmid pSLT. The pSLT-encoded SpvR negatively regulates an unknown repressor of the phenotype located on pSLT. Finally, disruption of carB leads to multiple auxotrophy that prevents lgp-tubule formation. This study demonstrates that lgp-tubule formation is a virulence mechanism that underlies the selective disruption of host endocytic trafficking and is associated with the formation of a replication-permissive locale.     

Taking possession: biogenesis of the Salmonella-containing vacuole

The Gram-negative pathogen Salmonella enterica can survive and replicate within a variety of mammalian cells. Regardless of the cell type, internalized bacteria survive and replicate within the Salmonella -containing vacuole, the biogenesis of which is dependent on bacterially encoded virulence factors. In particular, Type III secretion systems translocate bacterial effector proteins into the eukaryotic cell where they can specifically interact with a variety of targets. Salmonella has two distinct Type III secretion systems that are believed to have completely different functions. The SPI2 system is induced intracellularly and is required for intracellular survival in macrophages; it plays no role in invasion but is categorized as being required for Salmonella -containing vacuole biogenesis. In contrast, the SPI1 Type III secretion system is induced extracellularly and is essential for invasion of nonphagocytic cells. Its role in post-invasion processes has not been well studied. Recent studies indicate that Salmonella -containing vacuole biogenesis may be more dependent on SPI1 than previously believed. Other non-SPI2 virulence factors and the host cell itself may play critical roles in determining the intracellular environment of this facultative intracellular pathogen. In this review we discuss the recent advances in determining the mechanisms by which Salmonella regulate Salmonella -containing vacuole biogenesis and the implications of these findings.     

Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.     

Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.     

SpiC is required for translocation of Salmonella pathogenicity island 2 effectors and secretion of translocon proteins SseB and SseC

The Salmonella pathogenicity island 2 (SPI2) type III secretion system (TTSS) promotes Salmonella enterica serovar Typhimurium virulence for mice and increased survival and replication within eukaryotic cells. After phagocytosis, Salmonella serovar Typhimurium assembles the SPI2 TTSS to translocate over a dozen effector proteins across the phagosome membrane. SpiC has been previously shown to be a translocated effector with a large contribution to virulence (K. Uchiya, M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman, EMBO J. 18:3924-3933, 1999). This report demonstrates by competitive index that the virulence phenotype of a spiC mutant is equivalent to that of a secretion component mutant. In addition, translocation of SPI2 effector proteins was shown to require SpiC. Thus, the severe virulence phenotype resulting from deletion of spiC is likely due to the inability to translocate all SPI2 effectors. SpiC was also required to secrete translocon proteins SseB and SseC but not translocated effector SseJ, indicating that lack of assembly of the translocon explains the spiC mutant phenotype.     

The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors

Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative γ-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires' disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.     

Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor alpha-stimulated macrophages

Legionella pneumophila enhC ^- mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the Δ enhC strain, while tumour necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella- containing vacuole in TNF-α-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the Δ enhC mutant was more susceptible to H₂O₂ treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.     

Effector proteins encoded by Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton after translocation into host cells

The facultative intracellular pathogen Salmonella enterica has evolved strategies to modify its fate inside host cells. One key virulence factor for the intracellular pathogenesis is the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2). We have previously described SPI2-encoded SseF and SseG as effector proteins that are translocated by intracellular Salmonella . Detailed analysis of the subcellular localization of SseF and SseG within the host cell indicated that these effector proteins are associated with endosomal membranes as well as with microtubules. Specific association with microtubules was observed after translocation by intracellular Salmonella as well as after expression by transfection vectors. In epithelial cells infected with Salmonella , both SseF and SseG are required for the aggregation of endosomal compartments along microtubules and to induce the formation of massive bundles of microtubules. These observations demonstrate that SPI2 effectors interfere with the microtubule cytoskeleton and suggest that microtubule-dependent host cell functions such as vesicle transport or organelle positioning are altered by intracellular Salmonella .     

Internalization of Bordetella pertussis adenylate cyclase–haemolysin into endocytic vesicles contributes to macrophage cytotoxicity

Bordetella pertussis adenylate cyclase–haemolysin is a critical virulence factor in the murine model of intranasal infection, where it is required for several pathological effects, including macrophage apoptosis. Based on biochemical and immunological properties, it was proposed that the toxin was delivered directly to the cytoplasm of eukaryotic cells without trafficking through the endocytic pathway. In the present study, we analysed the cellular distribution of the adenylate cyclase–haemolysin during intoxication of macrophages. We showed that, shortly after its initial binding to the plasma membrane of macrophages, the toxin gains access to intracellular compartments that become progressively positive for the endosomal marker transferrin, but not for the lysosomal membrane protein CD107a/Lamp1. Importantly, the vesicular trafficking of the adenylate cyclase–haemolysin appears to be required for its ability to induce macrophage death. Inhibitors of actin polymerization and of macropinocytosis, as well as depletion of plasma membrane cholesterol and disruption of the Golgi network, reduce the toxin's ability to kill macrophages. Altogether, these results suggest that internalization of the adenylate cyclase–haemolysin into endocytic vesicles, at least partly through macropinocytosis, contributes to cytotoxicity.     

Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium

Background  

SpiC encoded within Salmonella pathogeniCity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.