Enterotoxigenic Escherichia coli CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions.

Colonization surface antigens (CSs) represent key virulence-associated factors of enterotoxigenic Escherichia coli (ETEC) strains. They are required for gut colonization, the first step of the diarrhoeal disease process induced by these bacteria. One of the most prevalent CSs is CS21, or longus, a type IV pili associated with bacterial self-aggregation, protection against environmental stresses, biofilm formation and adherence to epithelial cell lines. The objectives of this study were to assess the role of CS21 in adherence to primary intestinal epithelial cells and to determine if CS21 contributes to the pathogenesis of ETEC infection in vivo. We evaluated adherence of a CS21-expressing wild-type ETEC strain and an isogenic CS21-mutant strain to pig-derived intestinal cell lines. To determine the role of CS21 in pathogenesis we used the above ETEC strains in a neonatal mice challenge infection model to assess mortality. Quantitative adherence assays confirmed that ETEC adheres to primary intestinal epithelial cells lines in a CS21-dependent manner. In addition, the CS21-mediated ETEC adherence to cells was specific as purified LngA protein, the CS21 major subunit, competed for binding with the CS21-expressing ETEC while specific anti-LngA antibodies blocked adhesion to intestinal cells. Neonatal DBA/2 mice died after intra-stomach administration of CS21-expressing strains while lack of CS21 expression drastically reduced the virulence of the wild-type ETEC strain in this animal model. Collectively these results further support the role of CS21 during ETEC infection and add new evidence on its in vivo relevance in pathogenesis.

Mycoplasma pneumoniae-an emerging extra-pulmonary pathogen.

Mycoplasma is a well-recognised pathogen that colonises mucosal surfaces of humans and animals. Mycoplasma pneumoniae infects the upper and lower respiratory tracts of children and adults, leading to a wide range of respiratory and non-respiratory clinical conditions. M. pneumoniae infection is frequently considered in the differential diagnosis of patients with respiratory illnesses, and is commonly managed empirically with macrolides and fluoroquinolones. This contrasts with patients who present with non-respiratory symptoms in the context of a recent or current unrecognised M. pneumoniae infection, for whom this pathogen is rarely considered in the initial differential diagnosis. This review considers the microbiological, epidemiological, pathogenic and clinical features of this frequent pathogen that need to be considered in the differential diagnosis of respiratory and non-respiratory infections.

Longus pilus of enterotoxigenic Escherichia coli and its relatedness to other type-4 pili--a minireview.

Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili. These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E. coli and both longus and the colonization factor antigen III (CFA/III) of ETEC. These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements. While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids. Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them. This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC. Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively. This suggests that lngB may be an accessory gene involved in biogenesis of longus.

Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves alpha(v) integrin receptors.

Binding of vitronectin (VN) to Neisseria gonorrhoeae expressing the heparan sulfate proteoglycan (HSPG) specific Opa50 protein was recently shown to trigger bacterial internalization into distinct epithelial cell lines. We have investigated the role of VN-binding integrin receptors and protein kinase C (PKC) in VN-triggered bacterial uptake. Blocking integrin function by RGDS peptides or by antibodies specific to alpha(v)beta5 or alpha(v)beta3 resulted in an abrogation of VN-triggered bacterial internalization. Moreover, inhibitors of PKC were found to block VN-triggered uptake. The essential role of alpha(v) integrins and the presumable involvement of PKC in VN-triggered gonococcal uptake are discussed.

Construction and immunogenicity in mice of attenuated Salmonella typhi expressing Plasmodium falciparum merozoite surface protein 1 (MSP-1) fused to tetanus toxin fragment C.

One strategy to develop a multi-antigen malaria vaccine is to employ live vectors to carry putative protective Plasmodium falciparum antigens to the immune system. The 19 kDa carboxyl terminus of P. falciparum merozoite surface protein 1 (MSP-1), which is essential for erythrocyte invasion and is a leading antigen for inclusion in a multivalent malaria vaccine, was genetically fused to fragment C of tetanus toxin and expressed within attenuated Salmonella typhi CVD 908. Under conditions in the bacterial cytoplasm, the fragment C-MSP-1 fusion did not form the epidermal growth factor (EGF)-like domains of MSP-1; monoclonal antibodies failed to recognize these conformational domains in immunoblots of non-denatured protein extracted from live vector sonicates. The MSP-1 was nevertheless immunogenic. One month following intranasal immunization of BALB/c mice with the live vector construct, four out of five mice exhibited > or =four-fold rises in anti-MSP-1 by ELISA (GMT=211); a single intranasal booster raised titers further (GMT=1280). Post-immunization sera recognized native MSP-1 on merozoites as determined by indirect immunofluorescence. These data encourage efforts to optimize MSP-1 expression in S. typhi (e.g. as a secreted protein), so that the EGF-like epitopes, presumably necessary for stimulating protective antibodies, can form.

A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) organisms produce a characteristic histopathology in intestinal epithelial cells called attaching and effacing lesions. The eaeA gene is associated with attaching and effacing lesions and encodes intimin, a 94-kDa outer membrane protein. A 60-MDa plasmid, pMAR2, is essential for full virulence of EPEC strain E2348/69 (O127:H6). We have cloned sequences from pMAR2 that increase expression of the chromosomal eaeA gene as shown by increased alkaline phosphatase activity of an eaeA::TnphoA gene fusion, increased expression of the intimin protein, and increased production of eaeA mRNA. These sequences are called per for plasmid-encoded regulator. pMAR2-cured JPN15 containing cloned per sequences adheres to HEp-2 cells in greater numbers than JPN15 carrying the plasmid vector only. The cloned per sequences contain four open reading frames (ORFs) which have been designated perA through perD. Only perC can by itself activate expression of eaeA::TnphoA, although the levels of alkaline phosphatase activity seen with this ORF alone are considerably lower than those seen when all four ORFs are present. The molecular sizes of polypeptides predicted from perA, perB, perC, and perD ORFs are 24, 14.8, 10.5, and 9.4 kDa, respectively. The PerA predicted protein shares homology with members of the AraC family of bacterial regulators, but PerB, PerC, and PerD have no striking homology with previously described prokaryotic proteins. Our studies indicate that plasmid-encoded factors regulate the expression of eaeA and possibly genes encoding other outer membrane proteins and may be important for virulence of EPEC.

Motility mutants of Vibrio cholerae O1 have reduced adherence in vitro to human small intestinal epithelial cells as demonstrated by ELISA.

Vibrio cholerae must colonize the human small intestine to cause diarrhoeal disease. V.cholerae strains N16961 (EI Tor, Inaba) and 395 (classical, Ogawa) adhered to the epithelial cell surface and the mucus layer of isolated human small intestinal epithelial cells. They adhered specifically to the mucosa and apical membrane in thin sections of small intestine. No binding to the basolateral membrane of dissected epithelial tissue or to intracellular components of the epithelial cells was observed by either light or indirect immunofluorescence microscopy. Based on these results, a modified ELISA was developed to quantitatively study adherence of V. cholerae to human small intestinal epithelial cells. The assay used homogenized human small intestinal mucosal tissue as the substrate for binding. Treatment of the epithelial cell homogenate with 2-mercaptoethanol to disrupt protein and glycoprotein secondary structure inhibited the binding of V. cholerae strains, suggesting that binding was to specific receptors. Several V. cholerae strains and mutants from both biotypes were tested for adherence in the modified ELISA. Wild-type strains of both biotypes and non-enterotoxigenic strains, which were known to colonize humans, adhered. V. cholerae mutants defective in motility, flagellar structure of chemotaxis, which were known to exhibit reduced colonization in animal models, exhibited decreased adherence. The specificity of the assay and its ability to quantify binding should facilitate identification and the study of adherence factors involved in the colonization of human small intestinal epithelial cells by V. cholerae.

The attenuated Salmonella vaccine approach for the control of Helicobacter pylori-related diseases.

The Gram-negative bacterium Helicobacter pylori is a widespread human pathogen that colonizes the gastric mucosa and is associated with gastro-intestinal illnesses such as gastritis, peptic ulcer, gastric lymphoma and gastric cancer. Current pharmacological therapies are becoming less reliable for the control of H. pylori due to the elevated costs and to the increasing number of antibiotic resistant strains. New vaccination strategies utilizing H. pylori antigens combined with adjuvants or delivery of antigens by attenuated Salmonella strains have been successful in protecting mice against H. pylori infections. Oral immunization with single doses of urease-expressing Salmonella vaccine strains elicits mucosal and systemic antibody responses and fully protects different mouse strains against challenge infections with H. pylori. The high efficacy in the mouse model, combined with remarkable immunogenicity, safety and low-cost production, makes attenuated live recombinant Salmonella promising vaccine candidates for the control of H. pylori-related diseases in humans.

Expression, extracellular secretion, and immunogenicity of the Plasmodium falciparum sporozoite surface protein 2 in Salmonella vaccine strains.

Deleting transmembrane alpha-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04).

Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells.

Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections.

Ligation of cell surface heparan sulfate proteoglycans by antibody-coated beads stimulates phagocytic uptake into epithelial cells: a model for cellular invasion by Neisseria gonorrhoeae.

Binding of a particular opacity outer membrane protein (Opa) of Neisseria gonorrhoeae to cell surface heparan sulfate proteoglycans (HSPGs) of epithelial cells results in tight bacterial adherence; however, the role of this ligand-receptor interaction in triggering the subsequent bacterial internalization step is uncertain. Here we have used latex beads coated with HSPG-ligating antibodies as an in vitro model to study the role of HSPGs in gonococcal uptake into epithelial cells. Beads and gonococci showed the same cell line-specified adherence patterns and increase in phagocytic uptake mediated by serum or purified vitronectin (Vn). Heparitinase digestion as well as antibody competition experiments indicate that a critical level of HSPG ligation is necessary and sufficient to trigger phagocytic uptake into epithelial cells. Vn was found to specifically enhance HSPG-dependent phagocytic uptake while phagocytosis resulting from the ligation of other cell surface receptors was unaffected in the presence of Vn. Pharmacological studies with PKC inhibitors suggest a role for PKC in phagocytic uptake of HSPG-ligating beads. The use of drugs impairing cytoskeletal functions indicates that HSPG-dependent phagocytosis requires actin polymerization by a process distinct from receptor-mediated endocytosis.

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B.

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.

A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens.

The lack of a practical small animal model to study the immunogenicity of Salmonella typhi-based live vector vaccines expressing foreign antigens has seriously impeded the vaccine development process. For some foreign antigens, stimulation of serum IgG antibody is the desired, protective immune response. We administered to mice, by orogastric or intranasal (i.n.) routes, attenuated delta aroC delta aroD S. typhi CVD 908 carrying a plasmid encoding fragment C (fragC) of tetanus toxin fused to the eukaryotic cell receptor binding domain of diphtheria toxin (fragC-bDt), and monitored serum antibody. While orogastric inoculation of three doses was not immunogenic, i.n. immunization elicited high titers of serum IgG tetanus antitoxin, generating peak ELISA geometric mean titers (GMT) of 27024 and 35658 with 10(8) and 10(9) c.f.u. dosages, respectively; 10(9) c.f.u. i.n. of an delta aroA S. typhimurium live vector stimulated a peak antitoxin GMT of 376 405. Mice immunized with the S. typhi live vector were 100% protected against challenge with 100 50% lethal doses of tetanus toxin that rapidly killed all control mice. Intranasal immunization with two doses of S. typhi expressing unfused fragment C under control of an anaerobically-activated promoter derived from nirB stimulated significantly higher titers of serum neutralizing antitoxin than fused fragC-bDt controlled by the same promoter (GMT 0.10 AU ml-1 vs 0.01 AU ml-1, P = 0.0095). Two i.n. doses of S typhi encoding fragC under control of powerful constitutive promoter 1pp led to significantly higher peak serum neutralizing antitoxin titers than the otherwise identical construct utilizing the nirB promoter (peak GMT 0.72 AU ml-1 vs 0.10 AU ml-1, P = 0.022). The i.n. route of inoculation of mice may constitute a practical breakthrough that could expedite the development of some S. typhi-based live vector vaccines by allowing, for the first time, quantitative measurement of serum antibody responses to candidate constructs following i.n. mucosal immunization.