Alistair James Standish

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in all ages worldwide, and with ever-increasing antibiotic resistance, the understanding of its pathogenesis and spread is as important as ever. Recently, we reported the presence of a Low Molecular Weight Tyrosine Phosphatase (LMWPTP) Spd1837 in the pneumococcus. This protein is encoded in an operon, OM001 with two other genes, with previous work implicating this operon as important for pneumococcal virulence. Thus, we set out to investigate the role of the individual genes in the operon during pneumococcal pathogenesis. As LMWPTPs play a major role in capsular polysaccharide (CPS) biosynthesis in many bacteria, we tested the effect of mutating spd1837 and its adjacent genes, spd1836 and spd1838 on CPS levels. Our results suggest that individual deletion of the genes, including the LMWPTP, did not modulate CPS levels, in multiple conditions, and in different strain backgrounds. Following in vivo studies, Spd1836 was identified as a novel virulence factor during pneumococcal invasive disease, in both the lungs and blood, with this protein alone responsible for the effects of operon’s role in virulence. We also showed that a deletion in spd1836, spd1838 or the overall OM001 operon reduced survival in human saliva during the conditions that mimic transmission compared to the wildtype strain. With studies suggesting that survival in human saliva may be important for transmission, this study identifies Spd1836 and Spd1838 as transmission factors, potentially facilitating the spread of the pneumococcus from person to person. Overall, this study hopes to further our understanding of the bacterial transmission that precedes disease and outbreaks.

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae this transmembrane adaptor is CpsC, with the C-terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest CpsC has two transmembrane domains, with the N and C-termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal HA-tagged Cps2C protein in D39. Incubation of both protoplasts and membranes with the CP-B resulted in complete degradation of HA-Cps2C in all cases, indicating that the C-terminus of Cps2C was likely extra-cytoplasmic, and hence the protein's topology was not as predicted. Similar results were seen with membranes from TIGR4, indicating Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C-terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C-terminus by flow cytometry indicated that it was extra-cytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this affect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C-terminus flipping across the cytoplasmic membrane where it interacts with CpsD in order to regulate tyrosine kinase activity.

Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria.

Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both Gram-negative and –positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated Gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae ¸ which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.