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

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 infectionThesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 201

List of strains used.

<p>List of strains used.</p

Survival of D39 bacteria and mutant derivatives in human saliva.

<p>A starting concentration of 10⁶ CFU mL^-1 wildtype or mutant bacteria were incubated with saliva at the two conditions; (A) at 25°C without CO₂ and (B) at 37°C with CO₂. Experiments were performed in duplicate and repeated three times, independently. There was an approximately 0.5-log decrease in viable count for the D39 strain grown at 25°C without CO₂ and approximately 2-log decrease in viable count for the D39 strain grown at 37°C with CO₂. The CFU for D39 at t = 24 at 25°C without CO₂ was 5 × 10⁵ and the CFU for D39 at t = 24 at 37°C with CO₂ was 1 × 10⁴. Data were normalized such that the values represent the survival percentage of the mutant strains relative to the wildtype D39 (taken as 100%) ± SEM. Statistical differences between survival of <i>S</i>. <i>pneumoniae</i> in multiple dilutions of saliva were assessed by one-way ANOVA and Dunnett’s post hoc tests. **, P < 0.01, ***, P < 0.001.</p

Infection of mice with D39 bacteria and their mutant derivatives.

<p>Mice were challenged with D39, D39Δ<i>spd1837</i>, D39Spd1837_C8S, D39Δ<i>spd1838</i>, D39Δ<i>spd1836</i> and D39Δ<i>OM001</i>. Bacteria were enumerated from the nasopharynx and pleural lavage (A), lungs (B) and blood (C) of each mouse at 48 h post-infection (n = at least 5 per group). Horizontal line represents geometric mean; horizontal broken line denotes limit of detection abbreviated as LD (250 CFU for (A), 100 CFU for (B) and 50 CFU for (C)). *, P < 0.05; Statistical significance was calculated on log-transformed data using Mann Whitney tests. The incidence of pneumococcal invasion into the lungs and blood of mice were compared using two-tailed Fisher’s exact test.</p

Proteins encoded by <i>OM001</i> operon do not alter tyrosine phosphorylation of CpsD.

<p>Proteins from whole-cell lysates from D39, D39Δ<i>spd1837</i>, D39Spd1837_C8S, D39Δ<i>spd1838</i>, D39Δ<i>spd1836</i> and D39Δ<i>OM001</i> cells were separated by SDS-PAGE, and Western immunoblotting was undertaken with anti-Spd1837 (A), anti-CpsD (B), anti-phosphotyrosine (PY) (C), anti-CpsB (D) and anti-CbpA (E). MW, molecular weight (in kDa). The arrow on (C) indicates a band corresponds to CpsD.</p