Streptococcus pneumoniae resistance to cationic antimicrobial peptides: role of capsule and Pneumococcal surface protein A

Abstract

Streptococcus pneumoniae is an important pathogen responsible for high mortality rates worldwide. Vaccines used today against this pathogen are based on capsular polysaccharides, and they show limited coverage and high production costs, which limit its use in less developed regions. The pneumococcal surface protein A is an antigen widely studied as a possible vaccine candidate against pneumococcal infections; it shows high immunogenicity and provides protection in different animal models. One of the actions described for PspA involves the interaction with human lactoferrin, an antimicrobial protein found in milk and other secretions which releases bactericidal peptides (AMPs) upon proteolysis. The presence of PspA protects the bacterium from the lytic action of lactoferrin. Since lactoferrin's action is similar to that of other cationic antimicrobial peptides, this study aims to determine if PspAs protective effect includes other cationic AMPs, such as LL-37 and indolicidin - two CAMPs belonging to cathelicidin family. Initially, pneumococcal resistance to lysis by the peptides will be determined through an in vitro bactericidal assay. PspA's effect over CAMP-mediated pneumococcal killing will be investigated by comparing wild type and PspA-negative mutant strains. In order to determine the effect of free PspA over CAMP action, a competition assay will be performed by adding recombinant PspA fragments to the pneumococci-CAMP mix. The effect of anti-PspA antibodies to the lytic action of the peptides will also be evaluated. Direct interaction of PspA with the CAMPs will be analysed by western blot, using lactoferrin as a positive control. Finally, the contributions of polysaccharide capsule to bacterial resistance will be assessed by comparingLL-37 and indolicidin action on wild type and PspA-negative pneumococci with and without capsule. Additionally, the effect of serotype variations over CAMP action will be determined by comparing pneumococci of diverse serotypes. Taken together, the results will help elucidate PspA's role on protecting pneumococci against antimicrobial peptides, an important defense mechanism in the initial stages of bacterial pathogenesis. Furthermore, considering the immunogenic potential of PspA, the data from this study will aid on the development of safer and more efficient pneumococcal vaccines. (AU)