The Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2

Shigella flexneri is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of Shigella flexneri (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an in silico 3D model of OmpA from S. flexneri 5a strain M90T, we observed that the OmpA residues ₁₈₈EVQ₁₉₀ are likely essential for PhoN2-OmpA interaction. The ₁₈₈EVQ₁₉₀ amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction.     

The Periplasmic Enzyme,AnsB, of Shigella flexneri Modulates Bacterial Adherence to Host Epithelial Cells

S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.     

Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin-based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin-based motility. IcsA is inserted into the outer membrane as a 120-kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95-kDa amino-terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin-based motility, with a mean speed 27% faster than the wild-type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin-based motility of wild-type serotype 2a S. flexneri .     

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Effect of O side-chain length and composition on the virulence of Shigella flexneri 2a

IcsA of Shigella flexneri is required for intercellular spread and is located in the outer membrane at one pole of the bacterium, where it catalyses the polymerization of host-cell actin. The formation of the actin tail provides the force to move the bacterium in a unidirectional manner through the host-cell cytoplasm. We have previously demonstrated that rough lipopolysaccharide (LPS) mutants of S. flexneri 2a are avirulent and cannot form plaques in tissue-culture monolayers. This inability to form plaques is associated with non-polar localization of IcsA and loss of host-cell membrane-protrusion formation ('fireworks'). To define the minimal LPS structure required for fireworks formation, we constructed a strain of S. flexneri (BS497) that contains a mutation in rfc, encoding the O side-chain polymerase, and a strain, BS520, that possesses a defective O side-chain ligase due to a mutation in rfaL. BS497 produces a LPS that consists of a core with one repeat unit of the O side-chain, while BS520 produces a LPS consisting of a complete core with no O side-chain. BS497 remained invasive but did not form fireworks or plaques in tissue-culture monolayers and was negative in the Serény test. BS520 was invasive, generated reduced numbers of short fireworks, and made tiny plaques, but it was negative in the Serény test. Analysis of BS497 with anti-lcsA antibody demonstrated that IcsA was distributed over the entire cell surface. The distribution of IcsA on the surface of BS520 was predominantly unipolar, with some trail-back of IcsA label along the sides of the bacterium. A similar pattern was seen when infected monolayers were stained for polymerized actin. These results suggest that both the presence and the length of the O side-chain are important in the proper localization or maintenance of IcsA at the pole which subsequently affects the ability to form actin tails and produce fireworks. This reduced ability to form actin tails and fireworks results in a decreased ability of Shigella to move into adjacent host cells. To determine if the sugar composition of the O side-chain is important in the ability to form fireworks, the rfb region of S. flexneri2a was replaced with the rfb region from Escherichia coll serotype O8 or O25. Both hybrids were invasive, formed plaques, and gave positive Serény reactions. These results suggest that, unlike LPS length, the sugar composition of the O side-chain is not a critical requirement for the proper localization of IcsA and efficient intercellular movement.     

A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a

Background

Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE) analysis to measure changes in the expression profile that are induced by a temperature increase.

Results

The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgT_Y225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains.

Conclusion

Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.     

Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase,ERK and NF-κB

B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA) of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs). The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs), ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an optimal vaccine antigen.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a, induces protective immune response in a mouse model

Background

In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant.

Key Results

In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines.

Conclusion

These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.     

OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA_74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding.     

Connexin 26 facilitates gastrointestinal bacterial infection in vitro

Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.     

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

Shigella flexneri serotype 2a is a major public health concern in the developing and under-developed countries which contributes to shigellosis endemic and mortality. Thus, there is an urgent need for a rapid diagnostic test for effective therapy and disease management. Previous study showed that a ∼35 kDa antigenic protein from S. flexneri is a potential biomarker. We therefore modelled the three-dimensional structure of the antigen to probe its functionality which could aid in the development of an antigen-based diagnostic. Results showed that the antigen is a transmembrane protein consists of OmpA and OmpA-like domains. The OmpA domain is a beta-barrel embedded in the outer membrane with four surface-exposed extracellular loops. The OmpA-like domain is linked to the OmpA domain with a 17 amino acids linker and located in the periplasmic. Docking of peptidoglycan into the groove of OmpA-like domain might help in catalyzing the bacterial cell wall formation. Both domains are expected to be involved in the virulence, structural stability, pathogenesis and survival of Shigella thus made the 35 kDa protein a suitable shigellosis diagnostic biomarker. This structural elucidation will also enable a better identification of the epitope regions for the development of specific binders to the 35 kDa antigen.     

Phosphorylation of IcsA by cAMP-dependent protein kinase and its effect on intercellular spread of Shigella flexneri

Shigella flexneri, a Gram-negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120kDa bacterial outer membrane protein responsible for intracellular and cell-to-cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic-dependent protein kinases. Site-directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754–760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a ‘super Ics’ phenotype, characterized by an increased capacity to spread from cell-to-cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host-cell phosphorytation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during the invasion process.     

Contribution of the Periplasmic Chaperone Skp to Efficient Presentation of the Autotransporter IcsA on the Surface of Shigella flexneri

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.Type V secretion apparatuses (also called autotransporters) consist of an extensive class of large, outer membrane proteins of gram-negative bacteria, typically virulence factors, found in all subdivisions of proteobacteria (28). Although originally designated as “autotransporters” because they were thought to mediate their own insertion into and translocation across the outer membrane, more recent evidence suggests that autotransporter secretion and insertion requires the aid of accessory factors (21, 29). Secretion involves the insertion of the carboxy-terminal β-barrel domain into the outer membrane and translocation of the mature passenger (alpha) domain across the outer membrane (Fig. ​(Fig.1).1). Whether these two events occur sequentially or simultaneously is unclear. Analysis of crystal structures indicates that the carboxy-terminal end of the passenger domain is present within the central pore of the β-barrel (4, 27). Several studies provide evidence that at least some autotransporters are partially folded in the periplasm (7, 20), and one of these studies provides strong evidence that the passenger domain may be partially or fully incorporated into the β-barrel prior to incorporation of the mature protein into the outer membrane (20).Open in a separate windowFIG. 1.Schematic of the autotransporter IcsA. (A) Linear diagram showing the signal peptide (SP), alpha-domain (IcsA_53-757), and carboxy-terminal β-barrel domain. (B) IcsA in the outer membrane. The carboxy-terminal β-barrel is inserted into the outer membrane, and the mature amino-terminal alpha-domain is exposed on the bacterial surface. N′, mature amino terminus; C, carboxyl terminus; OM, outer membrane; arrow, proposed site of cleavage between residues 757 and 758 by IcsP.Shigella flexneri is a gram-negative human pathogen which, upon passage through the lower digestive tract, gains entry into colonic epithelial cells. Once S. flexneri is intracellular, it spreads to adjacent cells by secreting IcsA, a surface-associated autotransporter that is required for the polymerization of host cell actin on the bacterial surface. Actin polymerization occurs at a single pole of the bacterium and is required for infection of adjacent cells and disease pathogenesis (5, 24, 33).IcsA is encoded on a large virulence plasmid. The full-length protein is approximately 120 kDa and has three assigned functional and structural domains (25): an atypical Sec secretion signal (IcsA_1-52), the alpha-domain (IcsA_53-757), which is exposed on the bacterial surface and contains sequences that are required for actin polymerization, and the beta-domain (IcsA_758-1102), which forms a β-barrel structure in the outer membrane (Fig. ​(Fig.1A)1A) (21, 25). In vivo, a fraction of IcsA molecules are proteolytically processed at the junction between the alpha- and beta-domains by the protease IcsP (SopA), a protein which is also encoded on the virulence plasmid (14, 34). IcsA_53-757 is found in the supernatant of liquid cultures, while mature full-length IcsA (IcsA_53-1102), IcsA_758-1102 (14, 34), and some IcsA_53-757 (this work) remain cell associated. IcsA, like other autotransporters, is secreted at the bacterial pole (22), the site at which actin tail assembly occurs. As it is for other β-barrel-containing outer membrane proteins, insertion of IcsA and other autotransporters into the outer membrane requires the outer membrane insertase YaeT (BamA, Omp85) (21).Skp, DegP, and SurA are periplasmic chaperones that, like YaeT, appear to function in the targeting and/or insertion of outer membrane proteins (35). Evidence based on synthetic phenotypes suggests that during outer membrane protein insertion Skp and DegP act in one pathway and that SurA acts in a distinct but parallel pathway (35).We investigated the role of the periplasmic chaperone Skp in the folding and secretion of IcsA in S. flexneri. We found that in the absence of skp, IcsA is inefficiently presented on the surface of S. flexneri, leading to a cellular spread defect. Surprisingly, the protein was still efficiently cleaved by the outer membrane protease IcsP, as wild-type levels of IcsA_53-757 were detected in the culture supernatants. We found that introduction of the icsP mutation into the skp strain background led to an increase in the levels of full-length IcsA presented on the bacterial cell surface of the skp mutant, and we present models that could explain our results.     

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters in Shigella

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.The Shigella outer membrane protein IcsA is unusual in that it is secreted at the bacterial old pole (9, 13, 24). The secreted protein forms a cap at the old pole (Fig. ​(Fig.1A),1A), where during the infection of host cells, it interacts with cellular actin cytoskeletal proteins to induce the formation of propulsive actin tails (6, 43, 70). Actin tail formation is essential to the spread of Shigella spp. through cell monolayers and mammalian tissues (6, 43, 47) and is critical for Shigella virulence (15, 60). IcsA is a member of the autotransporter family of secreted proteins in gram-negative bacteria. Approximately 700 autotransporter proteins are predicted to be encoded within bacterial genomes that had been annotated as of 2003 (54). All autotransporter proteins for which the site of secretion has been determined are, like IcsA, secreted at the bacterial old pole (35).Open in a separate windowFIG. 1.Design of screen for proteins that, like IcsA, localize to potential division sites independent of FtsZ. (A) Localization of IcsA on the surface of S. flexneri. Immunofluorescence using antibody to IcsA. (B) Localization of IcsA_507-620-GFP (expressed from pBAD24-icsA_507-620::gfp) to poles of single cells of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the permissive temperature (30°C). (C) Localization of IcsA_507-620-GFP to potential cell division sites of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the nonpermissive temperature (42°C). (D) Diagram of the strategy used to identify proteins of E. coli that localize to potential cell division sites independent of FtsZ, displaying a localization pattern similar to that shown for IcsA in panel C. Image from DnaK-GFP localization (expressed from leaky promoter on pCA24N-dnaK, without induction) in screen well; incomplete overlay of GFP with phase-contrast microscopy is due to the movement of cells between capturing the two images, as cells were imaged live. Size bars = 2 μm (A and B) and 5 μm (C and D). Images are representative. O/N, overnight.Several other secreted bacterial proteins are also localized to one or both cell poles; these include the Listeria monocytogenes actin assembly protein ActA (39), components of the chemotaxis apparatus in Escherichia coli and Caulobacter crescentus (1, 46, 66), the Legionella pneumophila and Agrobacterium tumifaciens type IV secretion systems (14, 40), Pseudomonas aeruginosa type IV pili (8), protein components of the cell cycle regulatory pathways in C. crescentus (reviewed in reference 72), the DNA transfer apparatus in Bacillus subtilis and Streptomyces spp. (26, 28), and polar flagella in Vibrio cholerae, Campylobacter spp., Helicobacter spp., C. crescentus, and other gram-negative bacteria. In L. monocytogenes, the polarity of ActA is established after ActA secretion and likely depends on differential growth rates of the cell wall along the length of the bacterium (56). In C. crescentus, TipN serves as a polar developmental landmark (31, 42), and RcdA provides temporal and spatial specificity in the regulated proteolysis of key factors involved in polar asymmetry (50). Beyond these studies, relatively little is known about the molecular mechanisms that mediate the proper localization of polar bacterial proteins.Chemical or genetic blockade of cell division leads to the formation of filamentous cells without septa. In cells that have been filamented by either blocking FtsI or depleting functional FtsZ, a cytoplasmic derivative of IcsA localizes at or near potential cell division sites (36), which represent the sites of future cell poles. IcsA also localizes to potential division sites independent of nucleoid occlusion (36), together indicating that the positional information directing IcsA polarity is independent of these cell division proteins and chromosome positioning. The molecules that are required for the localization of IcsA to the cell pole have not been identified.One model of IcsA localization to the pole is that freely diffusing cytoplasmic IcsA recognizes and binds a protein receptor that is present at poles and future poles. Although icsA is present only in Shigella spp., upon heterologous expression, IcsA localizes to the poles of other Enterobacteriaceae (13, 58, 59), indicating that if targeting occurs via binding to a polar receptor, the receptor is likely conserved among members of this family. In addition, since IcsA localizes independently of FtsZ and FtsI, the localization of a putative polar receptor to the pole must also be independent of these cell division proteins. To find proteins that might serve as a polar receptor for IcsA, we first conducted a genome-wide screen designed to identify the subset of E. coli proteins that localize to poles and to potential cell division sites independently of functional FtsZ. For each conserved protein that displayed this localization pattern, we then tested whether it played a role in the polar localization of IcsA. We found that, under the conditions of our screen, a green fluorescent protein (GFP) fusion to the cytoplasmic chaperone DnaK localizes to cell poles and potential cell division sites. Although DnaK is not a polar receptor for IcsA, we demonstrated that it was required for the localization of IcsA to the pole in the bacterial cytoplasm in E. coli and for the secretion of both IcsA and a second Shigella autotransporter, SepA, in native Shigella flexneri, consistent with a critical role of DnaK in the chaperoning of IcsA and SepA, and perhaps autotransporter proteins more generally, in the bacterial cytoplasm.