Applying classical bacterial genetics to the discovery of a novel mechanism of antibody action against Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (pneumococcus) is the most common cause of community acquired pneumonia in the United States and globally. The ability of pneumococcus, a Gram positive bacterium, to cause disease in humans and experimental animals is governed by virulence factors, among which the most important is the pneumococcal capsular polysaccharide (PPS) that surrounds the bacterium (7). There are > 90 pneumococcal serotypes (ST), which are defined by uniquely repetitive structured PPS that are serologically distinct. Pneumococcal virulence has been attributed to the ability of the aforementioned polysaccharide capsules to inhibit host cell phagocytosis, but the question of why some STs are more virulent than others remains unresolved. The major goals of this thesis were: (i) to characterize the repertoire of protective anti-PPS binding antibody (Ab), (ii) to evaluate the contribution of capsules from strains that demonstrate virulence differences in their ability to induce lethal disease in mice, and iii) to investigate the effect of a MAb to PPS that does not promote host cell killing of pneumococcus in vitro on pneumococcal competence and quorum sensing.;In Chapter II, we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of ST8 (PPS8) cells and tetanus toxoid. Thirteen MAbs, including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC, were identified, and their nucleotide sequences, epitope and fine specificity, and efficacy against lethal challenge with ST8 pneumococcus were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germline variable region heavy (VH) and light (V L) chain genes with no evidence of somatic hypermutation. These findings reveal a relationship between MAb efficacy in mice and PPS specificity and VH gene use. The distinct gene use of protective MAbs to ST8 led us to delve deeper into the role of ST8 in pneumococcal virulence.;In Chapter III, we evaluated the contribution of the PPS of ST8, ST3 and ST2 to the virulence of these strains in mouse models of sepsis and pneumonia. We constructed 13 isogenic, capsule switched strains of ST2 (D39), ST3 (WU2, A66.1, and 6303), and ST8 (6308) and assessed their lethality in intraperitoneal and intranasal infection models in mice. Among the wild type (Wt) strains, using mouse death as the outcome of virulence, the order of virulence was ST8 > ST3 > ST2 in each infection model. However, we found that ST8 capsule expressing strains in the ST2 or ST3 background were avirulent. In contrast, ST3 capsule expressing strains in the ST2 background were more virulent than the Wt ST2 strain. Additionally, there were differences in the virulence of individual ST3 capsules (WU2, A66.1, and 6303) in this background. These findings demonstrate that ST3 capsular polysaccharide governs virulence and reveal previously unsuspected differences in the virulence of individual ST3 PPSs. Our data also show that the virulence of Wt ST8 is governed by additional factors.;Taken together, our data show that the pneumococcal capsules play distinct roles in virulence, induce Abs that use distinct gene elements and that certain capsule-binding Abs exert a direct effect on pneumococcal growth and biology by enhancing quorum sensing. The findings from this thesis provide insights that hold promise for the development of novel surrogates for pneumococcal vaccine efficacy and a roadmap for the development of next generation pneumococcal vaccines that could work by a direct effect on pneumococcal biology. (Abstract shortened by UMI.).