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.     

Cloning and transfer of the Salmonella pathogenicity island 2 type III secretion system for studies of a range of gram-negative genera

The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or "cassettes" of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.     

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.     

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.     

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.     

Cloning and Transfer of the Salmonella Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera

The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or “cassettes” of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.     

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 filamentous type III secretion translocon of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella , is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.     

InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium

The Salmonella effector protein SopA is translocated into host cells via the SPI-1 type III secretion system (TTSS) and contributes to enteric disease. We found that the chaperone InvB binds to SopA and slightly stabilizes it in the bacterial cytosol and that it is required for its transport via the SPI-1 TTSS.     

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1

Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.     

Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli

Type III secretion systems, designed to deliver effector proteins across the bacterial cell envelope and the plasma membrane of the target eukaryotic cell, are involved in subversion of eukaryotic cell functions in a variety of human, animal and plant pathogens. In enteropathogenic Escherichia coli (EPEC), several protein substrates for the secretion apparatus were identified, including EspA, EspB and EspD. EspA is a structural protein and the major component of a large transiently expressed filamentous surface organelle that forms a direct link between the bacterium and the host cell, whereas EspD and EspB seem to form the mature translocation pore. Recent studies of the type III secretion systems of Shigella and Salmonella pathogenicity island (SPI)-1 revealed the existence of a macromolecular complex that spans both bacterial membranes and consists of a basal structure with two upper and two lower rings and a needle-like projection that extends outwards from the bacterial surface. MxiH ( Shigella ) and PrgI ( Salmonella ) are the main components of the needle of the type III secretion complex. A needle-like complex has not yet been reported in EPEC. In this study, we investigated EscF, a protein sharing sequence similarity with MxiH and PrgI. We report that EscF is required for type III protein secretion and EspA filament assembly. Moreover, we show that EscF binds EspA, suggesting that EspA filaments are an extension of the type III secretion needle complexes in EPEC.     

Characterization of Salmonella type III secretion hyper-activity which results in biofilm-like cell aggregation

We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display.     

Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited

The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.     

EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.