In this study we demonstrate the role of human antibody directed to PhtD and PhtE to prevent adherence of pneumococcal to human NP epithelial cells. The results encourage further study of these antigens as vaccine ingredients in a third generation pneumococcal vaccine. Our experiments allowed the accumulation of several complimentary sets of data to justify our conclusions: (1) PhtD and PhtE-deficient TIGR 4 isogenic mutants were shown to bind to D562 and A549 cells significantly less well than the TIGR4 parent strain; (2) PhtD and PhtE when expressed individually on E coli
surface showed significantly enhanced adherence to the studied human respiratory epithelial cells; (3) The functional role of human antibody in preventing pneumococcal adherence to D562 cells was established by preparing human Fab fragments of antibody directed to the putative adhesins and showing that human adults Fabs directed to either PhtD or PhtE caused a direct effect of blockage of pneumococcal adherence to the human epithelial cells studied. The complementation of mutants is another method to determine whether a mutant phenotype can be reversed. We used another alternative approach - expression of an adhesin on the surface of heterologous host E coli
to corroborate the role of PhtD and PhtE as adhesins. The E coli
approach of expressing an adhesin on heterologous host surface has been described in the past by Chen et al
). However, in the absence of complementation we cannot be absolutely certain that the phenotypes observed in our mutants were a direct result of the mutation introduced. In addition, we demonstrated the functional role of anti-PhtD and anti-PhtE specific antibodies to block adherence of the pneumococci to human epithelial cells as a further validation of the role these proteins served as adhesins.
Extensive efforts are currently geared toward the development of effective alternative vaccination strategies against pneumococcal disease to address the shortcomings associated with capsule-based vaccines. Pneumococcal adherence to eukaryotic cells of the nasopharynx is a prerequisite for colonization and subsequent pathogenesis in the human host. These pathogen-host interactions are mediated by the binding of pneumococcal surface-exposed adhesins to specific cellular receptor molecules on human nasopharyngeal epithelial cells (29
). Pneumococcal colonization of the nasopharynx precedes the development of invasive disease and is the main gateway of transmission of the pathogen between individuals. Pneumococcal colonization is considered to be an immunizing event (32
), but the presence of natural antibodies may not be quantitatively or qualitatively adequate to prevent colonization. Adhesins from pneumococci and Non typeable Haemophilus influenzae
have been demonstrated to protect against infection by preventing the attachment of bacteria to host cells in animal models (6
) and could potentially translate in human upon vaccination. Antibodies directed against a pneumococcal adhesin protein might protect at the human mucosal surface by preventing pneumococcal attachment and subsequent colonization.
PhtD and PhtE are surface exposed, highly conserved proteins of the Pht family of pneumococcal proteins (21
). All four Pht proteins (Pht A, B, D and E) contain a classical lipoprotein motif (LxxC) within their N-terminal hydrophobic-leader sequences similar to that recognized for processing by signal peptidase II. PhtD and PhtE are 2520 and 3120 bp long open reading frames (ORF) and encode polypeptides of 839 and 1039 amino acids. PhtD and PhtE contain five and six histidine triad motifs and also contain segments that are predicted by colis algorithm to adopt coiled coil information, a feature common among gram positive surface proteins, as well as proline rich regions (14
). Signature tagged mutagenesis has suggested the involvement of these proteins in lung-specific virulence in mice (35
). Among their other putative roles, neutralization of complement factor C3b through factor H binding has been suggested (36
), though there are conflicting reports (17
). Proteins of the Pht family have been found to be protective against colonisation, pneumonia and sepsis in mouse models and the degree of protection offered was shown to be greater than other pneumococcal vaccine candidates including PspA and PsaA (37
). Isogenic mutants have shown a reduced binding in the case of pneumococcal PavA in the past but eventually it was found to be an indirect adhesin as PavA antisera could not reduce the binding of wild type pneumococci to cells (5
Until now, the role of human functional antibodies in preventing pneumococcal adherence has either been studied by using the serum or purified antibodies from serum (38
). Fabrizio et al
(2010) recently reported that pneumococcal polysaccharide specific human IgM leads to pneumococcal aggregation (26
). We also have recently found that pneumococci form bacterial aggregates when incubated with serum or purified IgG from human serum, thereby causing as impediment to pneumococcal adherence on host cells (Khan et al
under review). In nature an IgG mediated drop in pneumococcal adherence to host epithelial cells might be attributable completely to the aggregative effect of IgG on adherence since IgG mediated aggregation will facilitate clearance of the bacteria by cilia in the nasopharynx, sweeping the organisms towards the gastrointestinal tract. Although antibody-mediated pneumococcal aggregation could be a naturally occurring protective phenomenon during NP colonization, the role of functional antibodies directed against adhesin proteins can only be established in vitro by overcoming pneumococcal aggregation. Thus in this report we used IgG Fab fragments from purified serum IgG of adults and children to avoid the complication of formation of pneumococcal aggregates in the interpretation of the findings. Our results suggest the need of using IgG Fabs as a prerequisite to study the specific adhesin-blocking role of antibodies in pneumococcal adherence in vitro.
We found that adults raise functional adhesion-blocking antibodies against PhtD and PhtE proteins. Similar findings have been recently reported by Godfroid et al
(2011), where they have shown protection against pneumococcal colonisation in mice by vaccination with PhtD and PhtE against D39 pneumococci (37
). They have also demonstrated that the passive transfer of adult-raised PhtD antibodies can protect mice against lethal infection. In our experiments sera from children depleted of PhtD and PhtE specific Fabs did not result in a statistically significant increase in adherence. Given that the ELISA titers of the adults and children sera pools were quantitatively comparable for PhtD and PhtE, we interpret this finding to suggest that the functional ability of children antibodies in response to natural infection at an early age may not be similar to that of adults. The disparity in functional antibody between adults and children has been described in the past in various contexts as adult antibodies typically have greater affinity for antigen, and are more often bactericidal compared to children (39
Taken together, our findings clearly demonstrate that PhtD and PhtE are direct adhesins of Streptococcus pneumoniae, which elicit functional antibodies in adults. Further study of these antigens as vaccine candidates appears warranted.