Characterization of the Cell Wall Localization of Pneumolysin in Streptococcus pneumoniae.
bacterium Streptococcus pneumoniaeis a major causative agent of otitis media, pneumonia,
bacteremia and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent
cytolytic (CDC) pore-forming toxins, is produced by virtually all clinical isolates of
S. pneumoniae, and strains deleted for the pneumolysin gene are severely attenuated in
mouse models of colonization and i... read morenfection. In contrast to all other known members of
the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has
been hypothesized to be released upon autolysis, or alternatively, via a non-autolytic
mechanism that remains undefined. I show, using cell fractionation and Western blotting,
that exported Ply is localized primarily to the cell wall compartment in the absence of
detectable cell lysis. Hemolytic assays revealed that this cell wall-localized Ply is
active. Cell wall-localized Ply is accessible to extracellular protease and is detergent
releasable. Ply released by autolysis cannot re-associate with intact cells, suggesting
that there is indeed a Ply export mechanism that is coupled to cell wall localization of
the protein. Through truncation and domain swapping analyses, I show that export is
dependent on domain 2 of Ply. Additionally, a signal sequence is not sufficient for
Sec-dependent Ply secretion in S. pneumoniae, but is sufficient in a surrogate host
Bacillus subtilis. Finally, using the native ply sequence, I show that a signal
sequence-less protein export pathway is conserved in B.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Joan Mecsas, Michael Malamy, and Katya Heldwein.
Keyword: Microbiology.read less