| Capsular polysaccharide (CPS) production in the opportunistic pathogen Streptococcus pneumoniae (the pneumococcus) is a critical factor in the virulence of the pneumococcus during colonization and invasive infection. Modulation of CPS production is related to success in various host niches, and varying host environmental factors effect CPS modulation. Of particular interest are the effects of oxygen, manganese, and carbohydrates on CPS modulation. The biochemical mechanism of how such host environmental modulate CPS levels (thickness) is still relatively obscure. Such host environmental factors listed above may participate in regulating the enzymatic activity of CpsK, an enzyme critical to CPS production in most pneumococcal strains, due to its singular solvent exposed conserved active-site cysteine (Cys) at position 259. Some evidence in the literature indicates that such singular Cys-containing enzymes are prone to oxidation by reactive oxygen species, resulting in inhibition of enzyme activity. Manganese has been shown to protect Cys oxidation, thereby permitting Cys-containing enzymes remain active. We hypothesized that 1) oxidation of the active-site Cys would inhibit CpsK specific activity, and 2) that the presence of manganese would act as an antioxidant, thereby preserving CpsK specific activity. I show in vitro that the oxidation of Cys259 inhibits CpsK specific activity, providing a potential explanation for the observed effects of oxygen on CPS modulation. However, to our surprise, manganese treatment failed to protect oxidation of C259. Furthermore, extreme manganese concentrations above physiological levels decreased Cpsk specific
xi activity but to the extent of H2O2 treatment. Together, these data suggest that oxidants are capable of modulating CpsK specific activity. We provide several alternative plausible mechanisms for Mn in serving to indirectly protect and modulate CpsK activity in vivo, given the fluctuation of metal availability among various host environmental niches that the pneumococcus encounters. Key words: Streptococcus pneumoniae, capsular polysaccharide, manganese, hydrogen peroxide, UDP-glucose dehydrogenase, CpsK, 4-acetamido-4’-maleimidylstillbene-2,2’-disulfonic acid |