The importance of host-bacterial interactions in the pathogenesis of Streptococcus pneumoniae keratitis

Streptococcus pneumoniae (pneumococcus) is an important human pathogen capable of inducing a number of diseases including pneumonia, meningitis, otitis media and ocular infections. While the role of S. pneumoniae in other disease models is well characterized, its role in ocular diseases such as keratitis is not well studied. The aim of this study was to examine the relationship between bacterial and host strategies during S. pneumoniae keratitis infections.;Firstly this study aimed to elucidate the role Toll-like receptors (TLRs) play during pneumococcal keratitis, specifically TLR2 and TLR4. TLRs survey the extracellular environment and activate in the presence of molecular patterns found exclusively on pathogens. We compared the pathogenesis of the disease, bacterial viability, innate immune gene mRNA expression, and neutrophil invasion of C57BL/6 mice, as well as TLR2-/- and TLR4-/- mice in a C57BL/6 background. TLR2-/- mice exhibited higher quantities of viable bacteria, lower quantities of invading neutrophils, reduced innate immune response, and clinical damage scores similar to the C57BL/6 mice. In contrast, TLR4-/- mice exhibited higher quantities of invading neutrophils, similar quantities of viable bacteria to C57BL/6 mice, reduced innate immune response, and clinical damage scores significantly higher than the parent strain. TLR2 and TLR4 mRNA expression in the C57BL/6 cornea was verified with qRT-PCR at 72hrs while protein expression was verified using histological sections at both 24 and 72hrs post infection (p.i.).;Secondly, this study aimed to demonstrate that the pneumococcal toxin pneumolysin (PLY) activates TLR4 in a MyD88-independent manner. While all TLRs employ a MyD88-dependent signal cascade, TLR4 has an alternative MyD88-independent pathway. qRT-PCR data from infected C57BL/6 mice showed an upregulation in genes associated with the alternative, MyD88-independent pathway at both 24 and 72hrs p.i. Therefore, murine corneas were excised and treated with PLY or LPS and the protein concentrations of a MyD88-dependent cytokine (IL-6) and a MyD88-independent cytokine (RANTES) were determined. LPS induced both the MyD88- dependent and independent cytokine as the literature would suggest. PLY however only induced the MyD88-independent RANTES. Verification that PLY activates TLR4 independently of MyD88 was attempted using peptide inhibitors for both pathways but was unsuccessful due to the inhibitors themselves inducing IL-6 and RANTES. PLY induction of RANTES using corneas from a MyD88 -/- mouse model was abolished, casting doubt on its classification as a "MyD88-independent" cytokine.;Lastly, this study aimed to document internalization of pneumococcus in corneal cells during keratitis. Six keratitis clinical isolates were shown to bound and internalize corneal cells using an ex vivo corneal model. A capsule negative mutant, K1544ΔCAP, exhibited a significant increase in internalization and corneal damage compared to the parent K1544 strain. Addition of purified exogenous type 38 capsule reduced this internalization by 49% and significantly reduced the observed clinical damage at 24 and 48hrs p.i.;These studies highlight the importance of both bacterial and host actions during the progression of pneumococcal keratitis. While loss of innate immune signaling early in the infection may predispose the host to destructive corneal damage the presence or absence of key bacterial virulence factors can greatly impact the prognosis. An understanding of the relationship of both of these factors is critical to the development of novel and effective treatment regimens.