The related effector proteins SopD and SopD2 from Salmonella enterica serovar Typhimurium contribute to virulence during systemic infection of mice

Salmonella resides within host cells in a vacuole that it modifies through the action of virulence proteins called effectors. Here we examined the role of two related effectors, SopD and SopD2, in Salmonella pathogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) mutants lacking either sopD or sopD2 were attenuated for replication in the spleens of infected mice when competed against wild-type bacteria in mixed infection experiments. A double mutant lacking both effector genes did not display an additive attenuation of virulence in these experiments. The double mutant also competed equally with both of the single mutants. Deletion of either effector impaired bacterial replication in mouse macrophages but not human epithelial cells. Deletion of sopD2 impaired Salmonella's ability to form tubular membrane filaments [Salmonella-induced filaments (Sifs)] in infected cells; the number of Sifs decreased, whereas the number of pseudo-Sifs (thought to be a precursor of Sifs) was increased. Transfection of HeLa cells with the effector SifA induced the formation of Sif-like tubules and these were observed in greater size and number after co-transfection of SifA with SopD2. In infected cells, SifA and SopD2 were localized both to Sifs and to pseudo-Sifs. In contrast, deletion of sopD had no effect on Sif formation. Our results indicate that both SopD and SopD2 contribute to virulence in mice and suggest a functional relationship between these two proteins during systemic infection of the host.     

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis

Salmonella -induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin . We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.     

Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.     

Coiled-coil domains enhance the membrane association of Salmonella type III effectors

Coiled-coil domains in eukaryotic and prokaryotic proteins contribute to diverse structural and regulatory functions. Here we have used in silico analysis to predict which proteins in the proteome of the enteric pathogen, Salmonella enterica serovar Typhimurium, harbour coiled-coil domains. We found that coiled-coil domains are especially prevalent in virulence-associated proteins, including type III effectors. Using SopB as a model coiled-coil domain type III effector, we have investigated the role of this motif in various aspects of effector function including chaperone binding, secretion and translocation, protein stability, localization and biological activity. Compared with wild-type SopB, SopB coiled-coil mutants were unstable, both inside bacteria and after translocation into host cells. In addition, the putative coiled-coil domain was required for the efficient membrane association of SopB in host cells. Since many other Salmonella effectors were predicted to contain coiled-coil domains, we also investigated the role of this motif in their intracellular targeting in mammalian cells. Mutation of the predicted coiled-coil domains in PipB2, SseJ and SopD2 also eliminated their membrane localization in mammalian cells. These findings suggest that coiled-coil domains represent a common membrane-targeting determinant for Salmonella type III effectors.     

SopD acts cooperatively with SopB during Salmonella enterica serovar Typhimurium invasion

The intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), causes disease in a variety of hosts. To invade and replicate in host cells, these bacteria subvert host molecular machinery using bacterial proteins, called effectors, which they translocate into host cells using specialized protein delivery systems. One of these effectors, SopD, contributes to gastroenteritis, systemic virulence and persistence of S. typhimurium in animal models of infection. Recently, SopD has been implicated in invasion of polarized epithelial cells and here we investigate the features of SopD-mediated invasion. We show that SopD plays a role in membrane fission and macropinosome formation during S. typhimurium invasion, events previously shown to be mediated by the SopB effector. We further demonstrate that SopD acts cooperatively with SopB to promote these events during invasion. Using live cell imaging we show that a SopD-GFP fusion does not localize to HeLa cell cytosol as previously described, but instead is membrane associated. Upon S. typhimurium infection of these cells, SopD-GFP is recruited to the invasion site, and this recruitment required the phosphatase activity of SopB. Our findings demonstrate a role for SopD in manipulation of host-cell membrane during S. typhimurium invasion and reveal the nature of its cooperative action with SopB.     

Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.     

Analysis of the expression, secretion and translocation of the Salmonella enterica type III secretion system effector SteA

Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1-10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella.     

The Virulence Protein SopD2 Regulates Membrane Dynamics of Salmonella-Containing Vacuoles

Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA ⁻ mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA⁻ mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA⁻ mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA⁻ associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.     

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium . Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.     

SseF and SseG are translocated effectors of the type III secretion system of Salmonella pathogenicity island 2 that modulate aggregation of endosomal compartments

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI 2) is important for intracellular proliferation in infected host cells. Intracellular Salmonella use this system to translocate a set of effector proteins into the host cell. We studied the role of SseF and SseG, two SPI 2-encoded proteins. SseF and SseG are not required for translocation of effector proteins such as SseJ, encoded by genes outside of SPI 2. Rather, both proteins are translocated and interact with phagosomal membranes after translocation. In infected epithelial cells the formation of Salmonella-induced filaments, endosomal aggregates rich in lysosomal glycoproteins, is dependent on the function of SPI 2. We observed that, in mutant strains deficient for sseF or sseG, the formation of aggregated endosomes can take place, but the composition of the structures is different from those observed in cells infected with Salmonella wild type. These observations indicate that SseF and SseG modulate the aggregation of host endosomes.     

Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation

The Salmonella typhimurium type III secretion effector protein SifA is essential for inducing tubulation of the Salmonella phagosome and binds the mammalian kinesin-binding protein SKIP. Coexpression of SifA with the effector SseJ induced tubulation of mammalian cell endosomes, similar to that induced by Salmonella infection. Interestingly, GTP-bound RhoA, RhoB, and RhoC also induced endosomal tubulation when coexpressed with SseJ, indicating that SifA likely mimics or activates a RhoA family GTPase. The structure of SifA in complex with the PH domain of SKIP revealed that SifA has two distinct domains; the amino terminus binds SKIP, and the carboxyl terminus has a fold similar to SopE, a Salmonella effector with Rho GTPase guanine nucleotide exchange factor activity (GEF). Similar to GEFs, SifA interacted with GDP-bound RhoA, and purified SseJ and RhoA formed a protein complex, suggesting that SifA, SKIP, SseJ, and RhoA family GTPases cooperatively promote host membrane tubulation.     

SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg(2+) or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella.     

The first 45 amino acids of SopA are necessary for InvB binding and SPI-1 secretion

Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.     

Structural analysis of Salmonella enterica effector protein SopD

Salmonella outer protein D (SopD) is a type III secreted virulence effector protein from Salmonella enterica. Full-length SopD and SopD lacking 16 amino acids at the N-terminus (SopDDeltaN) have been expressed as fusions with GST in Escherichia coli, purified with a typical yield of 20-30 mg per litre of cell culture and crystallized. Biophysical characterization has been carried out mainly on SopDDeltaN. Analytical size exclusion chromatography shows that SopDDeltaN is monomeric and probably globular in aqueous solution. The secondary structure composition, calculated from the CD spectrum, is mixed (38% alpha-helix and 26% beta-strand). Sequence analysis indicates that SopD contains a coiled coil motif, as found in numerous other type III secretion system-associated proteins. This suggests that SopD has the potential for one or more heterotypic protein-protein interactions. Limited trypsin digestion of SopDDeltaN, monitored by both one-dimensional proton NMR spectroscopy and SDS-PAGE, shows that the protein has a large, protease-resistant core domain of 286 amino acid residues. This single-domain architecture suggests that SopD lacks a cognate chaperone. In crystallization trials, SopDDeltaN produced better crystals than either full-length SopD or trypsin-digested SopDDeltaN. Diffraction to 3.0 A resolution has so far been obtained from crystals of SopDDeltaN.     

The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.     

SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.

The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.     

The chaperone binding domain of SopE inhibits transport via flagellar and SPI-1 TTSS in the absence of InvB

Type III secretion systems (TTSS) are used by many Gram-negative pathogens for transporting effector proteins into eukaryotic host cells. Two modes of type III effector protein transport can be distinguished: transport into the surrounding medium (secretion) and cell-contact induced injection of effector proteins directly into the host cell cytosol (translocation). Two domains within the N-terminal regions of effector proteins determine the mode of transport. The amino terminal approximately 20 amino acids (N-terminal secretion signal, NSS) mediate secretion. In contrast, translocation generally requires the NSS, the adjacent approximately 100 amino acids (chaperone binding domain, CBD) and binding of the cognate chaperone to this CBD. TTSS are phylogenetically related to flagellar systems. Because both systems are expressed in Salmonella Typhimurium, correct effector protein transport involves at least two decisions: transport via the Salmonella pathogenicity island 1 (SPI-1) but not the flagellar TTSS (= specificity) and translocation into the host cell instead of secretion into the surrounding media (= transport mode). The mechanisms guiding these decisions are poorly understood. We have studied the S. Typhimurium effector protein SopE, which is specifically transported via the SPI-1 TTSS. Secretion and translocation strictly require the cognate chaperone InvB. Alanine replacement of amino acids 30-42 (and to some extent 44-54) abolished tight InvB binding, abolished translocation into the host cell and led to secretion of SopE via both, the flagellar and the SPI-1 TTSS. In clear contrast to wild-type SopE, secretion of SopE(Ala30-42) and SopE(Ala44-54) via the SPI-1 and the flagellar export system did not require InvB. These data reveal a novel function of the CBD: the CBD inhibits secretion of wild-type SopE via the flagellar and the SPI-1 TTSS in the absence of the chaperone InvB. Our data provide new insights into mechanisms ensuring specific effector protein transport by TTSS.     

Type III secretion by Salmonella typhimurium does not require contact with a eukaryotic host

The type III secretion system encoded by pathogenicity island I in Salmonella typhimurium delivers proteins to the external milieu and into the eukaryotic host cell. The principal factor in induction of the secretion system was found to be a change in the pH of the culture medium from acidic to mildly alkaline. The synthesis of components of the secretion machinery and the production and secretion of substrates occur simultaneously and do not require contact with a eukaryotic host cell. This argues against the concept that type III secretion in S. typhimurium is a process in which the delivery of a presynthesized pool of substrates is triggered by contact with a eukaryotic host cell.