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.     

pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI-2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI-2 type III secretion system. As the pH of the Salmonella-containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI-2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI-2-mediated secretion in vivo.     

Cloning of a functional Salmonella SPI-1 type III secretion system and development of a method to create mutations and epitope fusions in the cloned genes

Bacterial type III secretion systems have significant potential to be harnessed for beneficial purposes including vaccine development, anti-cancer therapies, strategies to counteract harmful bacteria-host interactions, and evolutionary studies. The ability to clone and manipulate type III secretion systems would allow researchers to perform novel experiments that would progress the biotechnological development of the potentially positive uses of these systems. Here, we report the cloning of the entire Salmonella pathogenicity island 1 (SPI-1) type III secretion system on a single DNA fragment that is contained on a self-transmissible plasmid vector for convenient transfer to alternate hosts. We demonstrate that the cloned SPI-1 type III system is functional for secretion and translocation via complementation of an S. typhimurium Delta SPI-1 strain. We also present a convenient method to construct mutations and epitope fusions in the cloned type III genes and demonstrate that the engineered substrate protein fusions are recognized by the cloned type III system. We transferred the cloned SPI-1 type III system into bacterial strains of different genera and found that there is a SPI-1 gene expression defect in these strains. The results describe a novel strategy for cloning and manipulation of bacterial secretion system gene clusters and provide a foundation for future studies to develop the beneficial uses of cloned type III secretion systems.     

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.     

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 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.     

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.     

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.     

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.     

Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella-containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.     

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.     

Salmonella-containing vacuoles: directing traffic and nesting to grow

Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative facultative intracellular pathogen that can infect a broad range of mammalian hosts. Following invasion of host cells, the majority of S. typhimurium are known to reside in a membrane-bound compartment known as the Salmonella-containing vacuole (SCV). S. typhimurium actively remodels this compartment using bacterial virulence proteins, called effectors, to establish a protected niche where it can replicate. S. typhimurium delivers more than 30 effectors into the host cell cytosol by bacterial type three secretion systems, encoded by Salmonella pathogenicity island 1 or 2 (SPI-1 or SPI-2). Recent studies have revealed a critical role for the SPI-1 effector SopB in 'directing traffic' at early stages of infection, allowing the bacteria to control SCV maturation by modulating its interaction with the endocytic system. At later stages of infection, the SCV establishes a 'nest' near the Golgi where optimal bacterial growth takes place. In this study, we highlight these recent developments in our understanding of SCV trafficking.     

Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These 'molecular syringes' inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.     

Identification of cognate host targets and specific ubiquitylation sites on the Salmonella SPI-1 effector SopB/SigD

Salmonella enterica is a bacterial pathogen responsible for enteritis and typhoid fever. Virulence is linked to two Salmonella pathogenicity islands (SPI-1 and SPI-2) on the bacterial chromosome, each of which encodes a type III secretion system. While both the SPI-1 and SPI-2 systems secrete an array of effectors into the host, relatively few host proteins have been identified as targets for their effects. Here we use stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics to identify the host targets of the SPI-1 effector, SopB/SigD. The only host protein found to bind immunoprecipitated SopB was the small G-protein Cdc42. The interaction was confirmed by reciprocal immunoprecipitation, and Cdc42 also bound glutathione S-transferase-fused SopB and SopB delivered through infection by the bacteria, confirming the interaction by an orthogonal method and in a more physiological context. The region of SopB responsible for the interaction was mapped to residues 117–168, and SopB is ubiquitylated at both K19 and K541, likely as monoubiquitylation. SopB colocalizes with activated Cdc42 near the plasmalemma, but we found no evidence that SopB alone can alter Cdc42 activity. This approach is also widely applicable to identify binding partners to other bacterial effectors.     

Salmonella SsrB activates a global regulon of horizontally acquired genes

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. This ability requires an S. enterica specific locus termed Salmonella pathogenicity island 2 (SPI-2). SPI-2 encodes a type III secretion system that injects effectors encoded within the island into host cell cytosol to promote virulence. SsrAB is a two-component regulator encoded within SPI-2 that was assumed to activate SPI-2 genes exclusively. Here, it is shown that SsrB in fact activates a global regulon. At least 10 genes outside SPI-2 are SsrB regulated within epithelial and macrophage cells. Nine of these 10 SsrB-regulated genes outside SPI-2 reside within previously undescribed regions of the Salmonella genome. Most share no sequence homology with current database entries. However, one is remarkably homologous to human glucosyl ceramidase, an enzyme involved in the ceramide signalling pathway. The SsrB regulon is modulated by the two-component regulatory systems PhoP/PhoQ and OmpR/EnvZ, and is upregulated in the intracellular microenvironment.     

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.