Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37.

Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log(10) CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log(10) CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.

Identification of immunodominant regions within the C-terminal cell binding domain of intimin alpha and intimin beta from enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains are a common cause of infantile diarrhea in developing countries. EPEC strains induce a characteristic attaching and effacing (A/E) lesion on epithelial cells. A/E lesion formation requires intimin, an outer membrane adhesin protein. The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of the polypeptide (Int280). So far, four distinct Int280 types (alpha, beta, gamma, and delta) have been identified. The aim of this study was to identify immunodominant regions within the Int280alpha and Int280beta domains. Recombinant DNA was used to construct and express overlapping polypeptides spanning these domains. Rabbit anti-Int280 antisera and human colostral immunoglobulin A were reacted with these polypeptides in Western blots and enzyme-linked immunosorbent assays. The results obtained with the rabbit antisera showed the presence of two separate immunodominant regions which are common to both Int280alpha and Int280beta. The first localized within the N-terminal region of Int280, and the second localized between amino acids 80 and 130. The results with the human colostra revealed one reactivity pattern against the Int280alpha fragments but two different reactivity patterns against the Int280beta domain.

Human colostrum contains IgA antibodies reactive to enteropathogenic Escherichia coli virulence-associated proteins: intimin, BfpA, EspA, and EspB.

BACKGROUND: In Brazil, enteropathogenic Escherichia coli diarrhoea is endemic among infants born into low economic levels, and it is one of the main causes of morbidity and mortality in this group. Binding of enteropathogenic E. coli to the brush border mucosa triggers a cascade of transmembrane and intracellular signals, causing cytoskeletal reorganization and formation of a specific lesion, termed the attaching and effacing lesion. Several enteropathogenic E. coli gene products have been implicated in formation of attaching and effacing lesions. Evaluation of pathogen-specific protective factors shows that breast feeding is effective against enteropathogenic E. coli infection. To investigate the nature of the protection, defatted colostrum and secretory immunoglobulin A obtained from mothers living in Sao Paulo were investigated for the ability to recognise selected enteropathogenic E. coli-associated virulence factors. METHODS: Western blot analysis was used to investigate the IgA repertoire in pooled colostrum that is reactive with specific enteropathogenic E. coli proteins. Whole enteropathogenic E. coli bacterial cell extracts, nonpathogenic E. coli strains overexpressing specific virulence factors, and purified polypeptides were used as antigen sources in this study. RESULTS: Reaction of the colostrum samples in Western blots of whole bacterial cell extracts and selected purified enteropathogenic E. coli proteins showed that they contained a secretory immunoglobulin A reactive with all the virulence-associated proteins studied. CONCLUSION: These results suggest that maternal antibodies may protect infants from enteropathogenic E. coli infection by interfering with adherence processes (anti-intimin and anti-bundle-forming pili antibodies) and cell signaling (anti-enteropathogenic Escherichia coli-secreted protein A and B antibodies.

Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

Down regulation of intimin expression during attaching and effacing enteropathogenic Escherichia coli adhesion.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.

Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.

A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.