Investigation of a low molecular weight protein tyrosine phosphatase in Streptococcus pneumoniae

Abstract

Tyrosine phosphorylation is a critical regulator of bacterial virulence, with the associated protein tyrosine phosphatases (PTPs) and bacterial tyrosine kinases (BY-kinases) recognised as major virulence factors in a range of bacterial pathogens including Streptococcus pneumoniae (the pneumococcus). The pneumococcus has a phosphoregulatory system comprising of CpsB (a PTP) and CpsC and CpsD, which together form an active BY-kinase. This system plays a crucial role in the regulation of the pathogen’s major virulence factor, the capsular polysaccharide (CPS). One open reading frame in the pneumococcal chromosome (designated spd1837) shows homology to the low molecular weight protein tyrosine phosphatases (LMWPTPs). LMWPTPs mediate CPS regulation in many other bacteria. Thus, investigating what role this protein plays in pneumococcal biology is the overreaching goal of this study. Purification of the phosphatase expressed in E. coli showed that Spd1837 was indeed a LMWPTP, with specificity against phosphotyrosine. spd1837 mutation was constructed on the chromosome of the pneumococcus and it was found that Spd1837 does not play a role in the regulation of CPS. The use of substrate-trapping assays, demonstrated that the phosphatase may interact with a variety of metabolic enzymes such as ATP-dependent-6-phosphofructokinase and Hpr kinase/phosphorylase, suggesting that the phosphatase may have roles in pneumococcal metabolism. In the chromosome of approximately 90% of pneumococcal strains with available genome sequence, spd1837 is co-transcribed together in the OM001 operon with the upstream translocase subunit YajC (Spd1838), and a downstream hypothetical protein (Spd1836). The OM001 operon was previously implicated to be important for pneumococcal virulence in a number of in vivo models. Here, we found that Spd1836 was essential for the bacterial ability to cause invasive disease in an established mouse model. Additionally, a previous genome-wide screen identified the OM001 operon to be important for pneumococcal growth and survival in human saliva. The data collected from this study suggest that human saliva can support the survival of the wildtype pneumococcal strain but not the mutant strain that carries a chromosomal deletions in spd1836 and spd1838 The pneumococcus is known to produce large quantities of hydrogen peroxide (H₂O₂) predominantly via the pyruvate oxidase, SpxB. It was found that the phosphatase activity of Spd1837 could be inhibited by H₂O₂ in vitro and Spd1837 itself confers protection against killing by H₂O₂. Whether SpxB played a role in regulating the activity of Spd1837 was then further investigated. Interestingly, in SpxB-deficient backgrounds and under aerobic conditions, Spd1837 modulated CPS biosynthesis, with Δspd1837ΔspxB and Spd1837C8SΔspxB [C8S subscript] showing significantly reduced CPS relative to both the wildtype and the ΔspxB strains. Therefore, the phosphatase Spd1837 does play a role in the pneumococcal CPS biosynthesis in an SpxB-dependent manner. The outcomes of this thesis highlight the importance of a number of previously unknown and uncharacterised bacterial factors during different stages of pneumococcal pathogenesis. Such research is critical to identify novel targets for anti-microbials against pneumococcal infection