Essential PcsB chap domain protein interacts with the essential FtsXSpn cell division protein in Streptococcus pneumoniae

During bacterial cell division, peptidoglycan (PG) is remodeled by precise hydrolysis and synthesis to allow cell constriction and daughter cell separation without lysing the cell. However, how bacteria couple PG remodeling to cell division remains poorly understood particularly in ovococcus bacteria such as human respiratory pathogen Streptococcus pneumoniae. In S. pneumoniae, PG homeostasis is regulated by the WalRK (VicRK/YycFG) two-component system, which positively regulates the expression of the essential pcsB gene. PcsB is a putative CHAP domain PG hydrolase, but purified PcsB lacks enzymatic activity in vitro. This leads to the hypothesis that PcsB is regulated by an additional unidentified component. My dissertation focuses on determining PcsB function by looking at the subcellular localization and the protein-protein interacting partners of PcsB. I showed by biochemical fractionation that other than being secreted, a significant portion of PcsB remain bound on the outer surface of the cell membrane by hydrophobic interaction. Consistent with a PG hydrolase involved in cell division, immunofluorescent microscopy reveals that bound PcsB is localized to the septal and equatorial region of dividing cells. By crosslinking and immunoprecipitation, I showed that PcsB interacts with the cell division protein complex FtsEX, which structurally resemble an ABC transporter. Both membrane protein FtsX and cytoplasmic ATPase FtsE are essential in pneumococcus. Cells depleted with FtsE and FtsX showed strikingly similar cell division defects comparing to the cells depleted of PcsB, suggesting FtsE, FtsX and PcsB are in the same biological pathway. Depleting FtsX will cause PcsB to delocalize and eventually release from the cells. Therefore PcsB is anchored to the cell surface by interacting with FtsX. In addition, mutations of the conserved residues required for FtsE ATPase activity are lethal. Also, I used genetic approaches to dissect the FtsEX:PcsB system to confirm the interaction between FtsX and PcsB. Suppressor mutations were identified on FtsX that rescue temperature sensitive (Ts) PcsB mutants. Interestingly, these suppressor mutations are located on the first and second extracellular loop of FtsX (ECL1 and ECL2), supporting the interaction between FtsX and the extracellular PcsB. I showed that pneumococcus is extremely susceptible to chemokine CXCL10 killing. Consistent with a previous report that CXCL10 targets FtsX in Bacillus anthracis, CXCL10 resistant mutants can be isolated by introducing mutations on FtsX. Together, these data support the idea that during cell division, FtsEX activates the PG hydrolytic activity of PcsB and couples it to cell division.