An investigation of the role that peptidoglycan composition and remodeling plays in the release of pneumolysin from Streptococcus pneumoniae.
pneumoniae (the pneumococcus) is a Gram-positive bacterium that asymptomatically
colonizes the human nasopharynx and is also a causative agent of several invasive
diseases. Pneumolysin (Ply) is a conserved virulence factor belonging to the
cholesterol-dependent cytolysin family of pore-forming toxins that is necessary for both
commensal and pathogenic lifestyles of this ... read moreorganism. Unlike all Gram-positive
cholesterol-dependent cytolysins characterized to date, Ply lacks a canonical
Sec-dependent signal sequence for export from the bacterial cell. Despite this, Ply
localizes to the cell wall compartment during growth, a feat that is conserved upon
expression in the heterologous host Bacillus subtilis, suggesting the existence of an
uncharacterized export pathway for leaderless extracellular proteins in Gram-positive
organisms. A genetic screen was performed to identify factors involved in Ply export in
B. subtilis. The results suggested an association between Ply export and cell wall
metabolism, which is the primary focus of my thesis. Working in the pneumococcus, I have
used a combination of subcellular fractionation, Western blot analysis and hemolysis
assays, to show that Ply release from the cell is inhibited by native peptidoglycan (PG)
structure. Mutation of the murMN operon responsible for branched stem peptide synthesis
revealed an inverse correlation between the abundance of this feature of PG and Ply
release. This relationship could partly be attributed to surface-associated
choline-binding proteins, which contribute to Ply release but appear to be sensitive to
the incorporation of branched stem peptides. Intriguingly, loss of branched stem
peptides decreased virulence in a murine model of pneumonia in a Ply-dependent manner
suggesting that increased Ply release during infection can be detrimental. Finally, as
further support for the association between PG and Ply release, I have characterized
SP0107, previously of unknown function, as a putative PG lysin. Truncation and point
mutation analysis has revealed that SP0107 is a secreted protein capable of binding PG
and, upon overexpression, causes increased Ply release in a manner dependent on its
putative PG degradative activity. SP0107 expression and activity are also associated
with increased sensitivity to penicillin- and vancomycin-induced cell death, further
supporting a role for this protein in modifying the PG
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Ralph Isberg, Abraham Sonenshein, and John Leong.
Keyword: Microbiology.read less