Streptococcus pneumoniae is a gram-positive bacterial pathogen that causes diseases such as pneumonia, meningitis, bacteremia and middle ear infections. The major virulence factor of S. pneumoniae is its polysaccharide capsule. The capsule enables the organism to evade host defenses by providing protection against complement-mediated opsonophagocytosis in systemic sites and by allowing the organism to successfully colonize the nasopharynx. The nasopharynx is the natural reservoir of S. pneumoniae. In the nasopharynx, S. pneumoniae is in a highly aerated (oxidizing) environment, however when it has the opportunity to bypass host defenses and invade systemic sites, it reaches environments with low aeration (reduced environments). Capsule production seems to correlate with environmental conditions. In aeration, capsule production is decreased, but it increases with decreasing aeration. Being able to rapidly modulate capsule production is important to the survival of the organism, as it experiences environmental changes throughout the stages of colonization and infection. Modulation of capsule production is controlled in part by a phosphotyrosine regulatory system that is comprised of a membrane sensing protein (Cps2C), a kinase (Cps2D) and a phosphatase/kinase inhibitor (Cps2B). In this project, we propose that capsule production may also be modulated through the production of hydrogen peroxide (H2O2). Under aerobic growth conditions, S. pneumoniae produces H2O2, which diffuses from the cell and accumulates in the culture medium in large quantities (>1 mM). The pyruvate oxidase, SpxB, is a major source of H2O2 in S. pneumoniae. SpxB is an important virulence factor that enhances the bacterium's ability to cause invasive disease. The goal of this project is to evaluate the dual effects of SpxB and environmental conditions on capsule production in S. pneumoniae. We hypothesize that capsule production is regulated in part by the effects of H2O2 on capsule biosynthetic enzymes. Under reduced aeration and low H2O2, environmental conditions similar in invasive disease, capsule production is increased thereby allowing survival of the organism. Ultimately, through this project, we may identify a posttranslational mechanism by which S. pneumoniae modulates the production of capsule biosynthesis as it travels from the nasopharynx to systemic sites to cause disease.
The specific aims for this project are to: 1) Determine effects of H2O2 and spxB mutations on capsule production;2) Determine effects of spxB mutations and H2O2 on enzyme activities and oxidation states;and 3) Determine effects of spxB mutations on virulence in vivo.
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is also a major cause of sepsis, meningitis, and otitis media (middle ear infections). The mechanisms by which this bacterium causes systemic disease are not fully understood. Identifying the regulatory element(s) S. pneumoniae uses to modulate a major virulence factor, the capsular polysaccharide, will provide insights into how a commensal bacterium becomes pathogenic and may lead to new therapeutics that not only target all pneumococci but organisms that use similar mechanisms.
