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.     

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.     

SiiA and SiiB are novel type I secretion system subunits controlling SPI4‐mediated adhesion of Salmonella enterica

The giant non‐fimbrial adhesin SiiE is essential to establish intimate contact between Salmonella enterica and the apical surface of polarized epithelial cells. SiiE is secreted by a type I secretion system (T1SS) encoded by Salmonella Pathogenicity Island 4 (SPI4). We identified SiiA and SiiB as two regulatory proteins encoded by SPI4. Mutant strains in siiA or siiB still secrete SiiE, but are highly reduced in adhesion to, and invasion of polarized cells. SiiA and SiiB are inner membrane proteins with one and three transmembrane (TM) helices respectively. TM2 and TM3 of SiiB are similar to members of the ExbB/TolQ family, while the TM of SiiA is similar to MotB and a conserved aspartate residue in this TM is essential for SPI4‐encoded T1SS function. Co‐immunoprecipitation, bacterial two‐hybrid and FRET demonstrate homo‐ and heterotypic protein interactions for SiiA and SiiB. SiiB, but not SiiA also interacts with the SPI4‐T1SS ATPase SiiF. The integrity of the Walker A box in SiiF was required for SiiB–SiiF interactionand SiiF dimer formation. Based on these data, we describe SiiA and SiiB as new, exclusively virulence‐associated members of the Mot/Exb/Tol family of membrane proteins. Both proteins are involved in a novel mechanism of controlling SPI4‐T1SS‐dependent adhesion, most likely by formation of a proton‐conducting channel.     

The Salmonella enterica giant adhesin SiiE binds to polarized epithelial cells in a lectin‐like manner

The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island (SPI) 1‐encoded type III secretion system (T3SS) and the SPI4‐encoded giant non‐fimbrial adhesin SiiE. SiiE is a highly repetitive protein composed of 53 bacterial Ig (BIg) domains and mediates binding to the apical side of polarized epithelial cells. We analysed the binding properties of SiiE and observed lectin‐like activity. SiiE‐dependent cell invasion can be ablated by chemical or enzymatic deglycosylation. Lectin blockade experiments revealed that SiiE binding is specific for glycostructures with terminal N‐acetyl‐glucosamine (GlcNAc) and/or α 2,3‐linked sialic acid. In line with these data, we found that SiiE‐expressing Salmonella bind to the GlcNAc polymer chitin. Various recombinant SiiE fragments were analysed for host cell binding. We observed that C‐terminal portions of SiiE bind to the apical side of polarized cells and the intensity of binding increases with the number of BIg domains present in the recombinant proteins. Based on these results, we propose that SiiE mediates multiple interactions per molecule with glycoproteins and/or glycosylated phospholipids present in the apical membrane of polarized epithelial cells. Thisintimate binding enables the subsequent function of the SPI1‐T3SS, resulting in host cell invasion.     

Functional dissection of SseF, a type III effector protein involved in positioning the salmonella-containing vacuole

Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.     

Structural and functional characterization of SiiA,an auxiliary protein from the SPI4‐encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.     

Salmonella pathogenicity island 2

Systemic infections by Salmonella enterica, such as typhoid fever, are a significant threat to human health. Recent studies indicate that the function of a type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) is central for the ability of S. enterica to cause systemic infections and for intracellular pathogenesis. This review summarizes approaches leading to the identification of SPI2, the molecular genetics and evolution of SPI2, and the current understanding of the regulation of gene expression. Recent studies have indicated that SPI2 is used by intracellular Salmonella to actively modify functions of the host cells. The role of SPI2 during pathogenesis of salmonellosis and current models regarding function will be discussed.     

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.     

Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells

Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.     

PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system

Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.     

Acidic pH is required for the functional assembly of the type III secretion system encoded by Salmonella pathogenicity island 2

Salmonella enterica employs two type III secretion systems (T3SS) for interactions with host cells during pathogenesis. The T3SS encoded by Salmonella pathogenicity island 2 (SPI2) is required for the intracellular replication of Salmonella and the survival inside phagocytes. During growth in vitro, acidic pH is a signal that promotes secretion of proteins by this T3SS. We analyzed protein levels and subcellular localization of various T3SS subunits under in vitro conditions at acidic or neutral pH, inducing or ablating secretion, respectively. Growth at acidic pH resulted in higher levels of SsaC, a protein forming the outer membrane secretin, without increasing expression of the operon containing ssaC. Acidic pH also induced oligomerization of SsaC subunits, a prerequisite for a functional secretin pore. It has previously been described that environmental stimuli resembling the intraphagosomal habitat of Salmonella control the expression of SPI2 genes. Here we propose that such stimuli also modulate the assembly of a functional T3SS that is capable of translocation of effector proteins into the host cell.     

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

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

Functional dissection of translocon proteins of the <Emphasis Type="Italic">Salmonella</Emphasis> Pathogenicity Island 2-encoded type III secretion system

Background  

Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. Salmonella enterica is a facultative intracellular pathogen that uses the Salmonella Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the Salmonella-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.     

Divergent roles of Salmonella pathogenicity island 2 and metabolic traits during interaction of S. enterica serovar typhimurium with host cells

The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium (S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied.     

Salmonella enterica serotype Typhimurium MisL is an intestinal colonization factor that binds fibronectin

MisL is an autotransporter protein encoded by Salmonella pathogenicity island 3 (SPI3). To investigate the role of MisL in Salmonella enterica serotype Typhimurium (S. Typhimurium) pathogenesis, we characterized its function during infection of mice and identified a host receptor for this adhesin. In a mouse model of S. Typhimurium intestinal persistence, a misL mutant was shed with the faeces in significantly lower numbers than the wild type and was impaired in its ability to colonize the cecum. Previous studies have implicated binding of extracellular matrix proteins as a possible mechanism for S. Typhimurium intestinal persistence. A gluthathione-S-transferase (GST) fusion protein to the MisL passenger domain (GST-MisL(29-281)) was constructed to investigate binding to extracellular matrix proteins. In a solid-phase binding assay the purified GST-MisL(29-281) fusion protein bound to fibronectin and collagen IV, but not to collagen I. MisL expression was not detected by Western blot in S. Typhimurium grown under standard laboratory conditions. However, when expression of the cloned misL gene was driven by the Escherichia coli arabinose promoter, MisL could be detected in the S. Typhimurium outer membrane by Western blot and on the bacterial cell surface by flow cytometry. Expression of MisL enabled S. Typhimurium to bind fibronectin to its cell surface, resulting in attachment to fibronectin-coated glass slides and in increased invasiveness for human epithelial cells derived from colonic carcinoma (T84 cells). These data identify MisL as an extracellular matrix adhesin involved in intestinal colonization.     

FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.     

Occurrence and characteristic of P-like adhesin among Klebsiella strains

Colonisation and remaining of microorganism on mucus membrane of microorganism is tightly connected with adhesion mechanisms and determine the first step of physiological settlement of the organism or the first stage of clinically demonstrated infection. In Klebsiella rods there are known three types of fimbrial adhesins (type 1, 3 and KPF-28) and non-fimbrial adhesin CF29K. It is stated that Klebsiella strains adhesions are responsible for their adherence to the epithelial cells of both respiratory and urinary tracts and to intestine epithelium. The in vitro research affirmed Klebsiella rods adherence to protein matrix. The aim of our work was the establishment of character, receptor specificity and the appearance frequency of P-like called adhesin. The frequency of expression of P-like adhesin was estimated among 380 isolated from the patients strains on the basis of agglutinating methods. The amorphic character of P-like adhesin was proved using electron microscopy method. The isolation and purification of P-like protein with a help of affinity chromatography enabled to estimate the receptor specificity of the adhesin. The receptor specificity was established as similar to E.coli PapG adhesin.