Functional dissection of SiiE, a giant non-fimbrial adhesin of Salmonella enterica

Salmonella enterica deploys the giant non-fimbrial adhesin SiiE to adhere to the apical side of polarized epithelial cells. The establishment of close contact is a prerequisite for subsequent invasion mediated by translocation of effector proteins of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type III secretion system (T3SS). Although SiiE is secreted into the culture medium, the adhesin is retained on the bacterial envelope in the phase of highest bacterial invasiveness. To dissect the structural requirements for secretion, retention and adhesive properties, comprehensive deletional and functional analyses of various domains of SiiE were performed. We observed that β-sheet and coiled-coil domains in the N-terminal moiety of SiiE are required for the control of SiiE retention on the surface and co-ordinated release. These results indicate a novel molecular mechanism for the control of surface display of a T1SS-secreted adhesin that acts cooperatively with the SPI1-T3SS.     

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.     

Salmonella Pathogenicity Island 4 encodes a giant non-fimbrial adhesin and the cognate type 1 secretion system

Pathogenicity Islands play a major role in the pathogenesis of infections by Salmonella enterica. The molecular function of Salmonella Pathogenicity Island 4 (SPI4) is largely unknown, but recent work indicated a role of SPI4 for Salmonella pathogenesis in certain animal models. We analysed the virulence functions of SPI4 and observed that SPI4 is contributing to intestinal inflammation in a mouse model. On a cellular level, SPI4 mediates adhesion to epithelial cells. We demonstrate the function of SPI4-encoded proteins as a type I secretion system (T1SS) and identify SiiE as the substrate protein of the T1SS. SiiE is secreted into the culture medium but mediates contact-dependent adhesion to epithelial cell surfaces. SiiE is a very large non-fimbrial adhesin of 600 kDa and consists of 53 repeats of Ig domains. Our study describes the first T1SS-secreted protein that functions as a non-fimbrial adhesin in binding to eukaryotic cells. The SPI4-encoded T1SS and SiiE might functionally resemble the type I fimbrial adhesins.     

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.     

Identification of a pathogenicity island required for Salmonella enteropathogenicity

Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin , and presented evidence that SopB is translocated into eukaryotic cells via a sip -dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella -specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA₁^Ser ( serT ) on one side and copS / copR on the other. Thus, this Salmonella -specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA , pipB , pipC , pipD (pathogenicity island-encoded proteins) and orfX , in addition to sopB . The effect of mutations in pipA , pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.     

Tandem translation generates a chaperone for the Salmonella type III secretion system protein SsaQ

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQ(L)) composed of 322 amino acids and a shorter protein (SsaQ(S)) comprising the C-terminal 106 residues of SsaQ(L). SsaQ(L) is essential for SPI-2 T3SS function. Loss of SsaQ(S) impairs the function of the T3SS both ex vivo and in vivo. SsaQ(S) binds to its corresponding region within SsaQ(L) and stabilizes the larger protein. Therefore, SsaQ(L) function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.     

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.     

SifA,a type III secreted effector of Salmonella typhimurium,directs Salmonella-induced filament (Sif) formation along microtubules

A unique feature of Salmonella enterica serovar typhimurium ( S. typhimurium ) is its ability to enter into (invade) epithelial cells and elongate the vacuole it occupies into tubular structures called Salmonella -induced filaments (Sifs). This phenotype is dependent on SifA, a Salmonella virulence factor that requires the SPI-2-encoded type III secretion system for delivery into host cells. Previous attempts to study SifA and other type III secreted proteins have been limited by a lack of suitable reagents. We examined SifA function by expressing SifA with two internal hemagglutinin epitope tags. By employing subcellular fractionation techniques, we determined that translocated SifA was membrane associated in infected HeLa cells. Confocal microscopy revealed that SifA associated with the Salmonella vacuole and with Sifs. Our analysis also revealed that microtubules serve as a scaffold for Sifs, and that SifA colocalizes with microtubules at sites of interaction between lysosomal glycoprotein-containing vesicles and Sifs. Treatment with the microtubule inhibitor nocodazole blocked Sif formation but did not prevent SifA translocation into the Salmonella vacuole. While polymerized actin has been observed on Sifs, this phenotype was transient and did not play a role in promoting or maintaining Sif formation. Our findings demonstrate the essential role of microtubule dynamics in the formation of Sifs and the utility of this epitope tagging strategy for the study of bacterial type III secreted proteins.     

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.     

Characterisation of proteins extracted from the surface of Salmonella Typhimurium grown under SPI-2-inducing conditions by LC-ESI/MS/MS sequencing

Salmonella enterica has two pathogenicity islands encoding separate type three secretion systems (T3SS). Proteins secreted through these systems facilitate invasion and survival. After entry, Salmonella reside within a membrane bound vacuole, the Salmonella containing vacuole (SCV), where translocation of a second set of effectors by the Salmonella pathogenicity island 2 (SPI-2) T3SS is initiated. SPI-2 secretion in vitro can be induced by conditions that mimic the Salmonella containing vacuole. Utilising high-throughput mass spectrometry, we mapped the surface-attached proteome of S. Typhimurium SL1344 grown in vitro under SPI-2-inducing conditions and identified 108 proteins; using secretion signal prediction software, 43% of proteins identified contained a signal sequence. Of these proteins, 13 were known secreted effector proteins including SPI-2 effector proteins SseB, SseC, SseD, SseL, PipB2 and SteC, although surprisingly five were SPI-1 proteins, SipA, SipB, SipC, SipD and SopD, while 2 proteins SteA and SlrP are secreted by both T3SSs. This is the first in vitro study to demonstrate dual secretion of SPI-1 and SPI-2 proteins by S. Typhimurium and demonstrates the potential of high-throughput LC-ESI/MS/MS sequencing for the identification of novel proteins, providing a platform for subsequent comparative proteomic analysis, which should greatly assist understanding of the pathogenesis and inherent variation between serovars of Salmonella and ultimately help towards development of novel control strategies.     

Structural and Biochemical Characterization of SrcA,a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.     

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.     

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.     

Growth Phase-Regulated Induction of Salmonella-Induced Macrophage Apoptosis Correlates with Transient Expression of SPI-1 Genes

Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.     

Cooperation of Salmonella pathogenicity islands 1 and 4 is required to breach epithelial barriers

Invasion is an important microbial virulence strategy to overcome the barrier formed by polarized epithelial cells. Salmonella enterica is a food-borne pathogen that deploys a type III secretion system for the manipulation of the actin cytoskeleton and to trigger internalization into epithelial cells. Here we show that this function is not sufficient to enter polarized cells and report that penetration of epithelia from the luminal side requires both the type III secretion system and novel virulence functions conferred by Salmonella pathogenicity island 4. Salmonella pathogenicity island 4 encodes a type I secretion system for the giant non-fimbrial adhesin SiiE that mediates intimate contact of Salmonella to microvilli on the apical membrane. Mutant strains lacking SiiE fail to invade polarized cells, to destroy epithelial barrier functions and to efface the apical brush border. Deletion analyses of repetitive domains in SiiE indicate that the large size of the adhesin is of functional importance. Our observations demonstrate that efficient penetration of epithelial barriers requires the cooperative activity of two Salmonella pathogenicity islands encoding different secretion systems. These findings underline the role of the epithelial brush border and reveal a new mechanism used by bacterial pathogens to overcome this barrier.