Molecular bases of epithelial cell invasion by Shigella flexneri

The pathogenesis of shigellosis is characterized by the capacity of the causative microorganism, Shigella, to invade the epithelial cells that compose the mucosal surface of the colon in humans. The invasive process encompasses several steps which can be summarized as follows: entry of bacteria into epithelial cells involves signalling pathways that elicit a macropinocitic event. Upon contact with the cell surface, S. flexneri activates a Mxi/Spa secretory apparatus encoded by two operons comprising about 25 genes located on a large virulence plasmid of 220 kb. Through this specialized secretory apparatus, Ipa invasins are secreted, two of which (IpaB, 62 kDa and IpaC, 42 kDa) form a complex which is itself able to activate entry via its interaction with the host cell membrane. Interaction of this molecular complex with the cell surface elicits major rearrangements of the host cell cytoskeleton, essentially the polymerization of actin filaments that form bundles supporting the membrane projections which achieve bacterial entry. Active recruitment of the protooncogene pp 60^c-src has been demonstrated at the entry site with consequent phosphorylation of cortactin. Also, the small GTPase Rho is controlling the cascade of signals that allows elongation of actin filaments from initial nucleation foci underneath the cell membrane. The regulatory signals involved as well as the proteins recruited indicate that Shigella induces the formation of an adherence plaque at the cell surface in order to achieve entry. Once intracellular, the bacterium lyses its phagocytic vacuole, escapes into the cytoplasm and starts moving the inducing polar, directed polymerization of actin on its surface, due to the expression of IcsA, a 120 kDa outer membrane protein, which is localized at one pole of the microorganism, following cleavage by SopA, a plasmid-encoded surface protease. In the context of polarized epithelial cells, bacteria then reach the intermediate junction and engage their components, particularly the cadherins, to form a protrusion which is actively internalized by the adjacent cell. Bacteria then lyse the two membranes, reach the cytoplasmic compartment again, and resume actin-driven movement.     

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.     

IpaC from Shigella and SipC from Salmonella possess similar biochemical properties but are functionally distinct

Invasion plasmid antigen C (IpaC) is secreted via the type III secretion system (TTSS) of Shigella flexneri and serves as an essential effector molecule for epithelial cell invasion. The only homologue of IpaC identified thus far is Salmonella invasion protein C (SipC/SspC), which is essential for enterocyte invasion by Salmonella typhimurium. To explore the biochemical and functional relatedness of IpaC and SipC, recombinant derivatives of both proteins were purified so that their in vitro biochemical properties could be compared. Both proteins were found to: (i) enhance the entry of wild-type S. flexneri and S. typhimurium into cultured cells; (ii) interact with phospholipid membranes; and (iii) oligomerize in solution; however, IpaC appeared to be more efficient in carrying out several of the biochemical properties examined. Overall, the data indicate that purified IpaC and SipC are biochemically similar, although not identical with respect to their in vitro activities. To extend these observations, complementation analyses were conducted using S. flexneri SF621 and S. typhimurium SB220, neither of which is capable of invading epithelial cells because of non-polar null mutations in ipaC and sipC respectively. Interestingly, both ipaC and sipC restored invasiveness to SB220 whereas only ipaC restored invasiveness to SF621, suggesting that SipC lacks an activity possessed by IpaC. This functional difference is not at the level of secretion because IpaC and SipC are both secreted by SF621 and it does not appear to be because of SipC dependency on this native chaperone as coexpression of sipC and sicA in SF621 still failed to restore detectable invasiveness. Taken together, the data suggest that IpaC and SipC differ in either their ability to be translocated into host cells or in their function as effectors of host cell invasion. Because IpaB shares significant sequence homology with the YopB translocator of Yersinia species, the ability for IpaC and SipC to associate with this protein was explored as a potential indicator of translocation function. Both proteins were found to bind to purified IpaB with an apparent dissociation constant in the nanomolar range, suggesting that they may differ with respect to effector function. Interestingly, whereas SB220 expressing sipC behaved like wild-type Salmonella, in that it remained within its membrane-bound vacuole following entry into host cells, SB220 expressing ipaC was found in the cytoplasm of host cells. This observation indicates that IpaC and SipC are responsible for a major difference in the invasion strategies of Shigella and Salmonella, that is, they escape into the host cell cytoplasm. The implications of the role of each protein's biochemistry relative to its in vivo function is discussed.     

Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri

Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.     

Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness.

The invasion of colonic epithelial cells by Shigella, an early essential step for causing bacillary dysentery, is mediated by the IpaB, IpaC and IpaD proteins. Secretion of the Ipa proteins from Shigella requires functions encoded by the mxi and spa loci. In this study, we show that contact between the bacteria and epithelial cell triggers release of the Ipa proteins into the external medium, which results in a rapid decrease in levels of Ipa proteins presented on the cell surface. When the bacteria were used to infect polarized Caco-2 cells, release of Ipa proteins occurred efficiently from bacteria interacting with the basolateral surface rather than with the apical surface. Moreover, the interaction of bacteria with components of the extracellular matrix, such as fibronectin, laminin or collagen type IV, also stimulates the release of Ipa proteins. The release of Ipa proteins from Shigella required the surface-located Spa32 protein encoded by one of the spa genes on the large plasmid.     

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1‐encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin–Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F‐actin in tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin‐stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild‐type and sipA mutant strains of S. typhimurium. Time‐lapse phase‐contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild‐type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild‐type bacteria or failed to invade at all. Furthermore, although wild‐type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild‐type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.     

Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Shigella flexneriand related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems fromS. flexneriinvasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid ofS. flexnerithat have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of theipaoperon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailedin vitroanalysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins fromEscherichia colifor use in dissecting of the protein biochemistry ofS. flexneripathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response toS. flexneriinvasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasion, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.     

E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9‐dependent mechanism

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3‐secreted effectors can interfere with IEC signalling pathways via specific protein–protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9‐induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non‐invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco‐2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane‐deforming activity of SNX9.     

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA supercoiling.     

Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.     

Significance of the secreted form of IpaC, a 45 kDa protein of Shigella dysenteriae 1, in the invasive process as determined by monoclonal antibodies

Abstract Invasion plasmid antigen C (IpaC), a 45 kDa plasmid encoded protein, is associated with the virulence of virulent Shigella spp. In S. dysenteriae type 1 the 45 kDa IpaC protein is secreted to a greater extent into the surrounding medium in comparison to other Shigella spp. Monoclonal antibodies (mAbs) to the secreted form of IpaC protein were raised in this study. Of the four secretory hybrid cells, one (3G4) was found to have a very high antibody titre as determined by ELISA. The specificity of 3G4 was confirmed by immunoblotting of whole cell extract of Escherichia coli strain MC1061 carrying the plasmid pHW756 which synthesizes both the IpaB and C proteins. The effect of the mAbs on plaque formation by virulent Shigella dysenteriae 1 was determined and it was found that the clone 3G4 substantially (55%) reduced plaque formation on HeLa cell monolayer. The epitope specificity of the mAb 3G4 was competitively inhibited by the convalescent phase sera from human, suggesting that the epitope recognized by clone 3G4 was expressed during the natural course of infection and also indicating that the 45 kDa (IpaC) protein in secreted form has a definite role in the invasive process.     

EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea. EPEC mediates several effects on host epithelial cells, including activation of signal-transduction pathways, cytoskeletal rearrangement along with pedestal and attachingleffacing lesion formation. It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria. DNA-sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino-terminal sequence of a 25 kDa EPEC secreted protein. A mutant with a non-polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. These phenotypes were complemented by a cloned espA gene. The espA mutant is also defective for invasion. It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.     

Expression of His-tagged Shigella IpaC in Arabidopsis

Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge. Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana. S. flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells. IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector. This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed. T1 and T2 plant generations were obtained. Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations. A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants. Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants. Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.     

FadA from Fusobacterium nucleatum utilizes both secreted and nonsecreted forms for functional oligomerization for attachment and invasion of host cells

Fusobacterium nucleatum is a Gram-negative anaerobe associated with various human infections, including periodontal diseases and preterm birth. A novel FadA adhesin was recently identified for host-cell binding. It consists of 129 amino acid residues, with an 18-amino acid signal peptide. Expression of FadA in Escherichia coli enhanced bacterial binding to host epithelial and endothelial cells. In both E. coli and F. nucleatum, FadA exists in two forms, the intact pre-FadA and the secreted mature FadA (mFadA), with pre-FadA anchored in the inner membrane and mFadA secreted outside the bacteria. Pre-FadA and mFadA formed high M(r) complexes. When each form was purified to a single species, mFadA was soluble at neutral pH, whereas pre-FadA was insoluble. Pre-FadA became soluble when mixed with mFadA or under acidic pH. When fluorescence-labeled mFadA alone was added to the epithelial cells, no binding was detected. However, when mixed with nonlabeled pre-FadA, binding and invasion of mFadA into epithelial cells was observed. FadA is a unique bacterial adhesin/invasin in that it utilizes its own two forms for both structural and functional purposes. The pre-FadA-mFadA complex is probably anchored in the inner membrane and protrudes through the outer membrane. Internalization of the pre-FadA-mFadA ensures invasion of the bacteria into the host cells.     

The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

The Sbi protein of Staphylococcus aureus comprises two IgG‐binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C‐terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram‐positive bacteria. Cell‐associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C‐terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA‐defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane.     

Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes

Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes.     

Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri

Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261-363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.     

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin‐binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin‐binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3‐Kinase, c‐Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1‐specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.     

The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.     

Identification and characterization of the mammalian association and actin-nucleating domains in the Rickettsia conorii autotransporter protein, Sca2

Establishment of infection by spotted fever group rickettsial species is dependent on the ability of these bacteria to adhere to and invade the host endothelium. Recent studies have attributed these processes to a handful of rickettsial surface proteins from the surface cell antigen (sca) family of autotransporters. A rickettsial autotransporter from Rickettsia conorii, Sca2, has been shown to be sufficient to mediate both adherence and invasion of human endothelial cells and to participate in intracellular actin‐based motility. Here we identify a region of Sca2 capable of interacting with the mammalian cell surface and show that this function of Sca2 is independent and separable from its actin nucleation activity. Furthermore, pre‐incubation of mammalian cells with the Sca2 mammalian association region prior to R. conorii infection can competitively inhibit rickettsial invasion, suggesting that Sca2 plays an important role in the initial interaction with mammalian cells. Together, our results demonstrate that the Sca2 autotransporter protein in R. conorii contains distinct functional domains that likely are involved in mediating cellular interactions at the plasma membrane and the host cytosol.