Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

9-4-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Michael G Caparon

Abstract

Streptococcus pyogenes and other Gram-positive pathogens are highly dependent on secreted virulence proteins for their ability to colonize a host and evade the immune effectors that act on pathogens to prevent this. Unlike eukaryotes and Gram-negative bacteria, all Gram-positive pathogens lack a cellular compartment dedicated to folding and processing secreted proteins once they have been translocated across the cytoplasmic membrane. S. pyogenes and other Gram-positive organisms overcome this challenge by clustering the secretory translocons at a defined anionic lipid microdomain of the cytoplasmic membrane that is enriched for accessory factors needed for protein biogenesis, referred to as the ExPortal. The manner in which S. pyogenes achieves and maintains spatial coordination of the factors that constitute the ExPortal is not understood. Given the importance of secreted virulence proteins to streptococcal pathogenicity, the ExPortal is an attractive target for the development of therapeutics that can efficiently counter streptococcal infections. I investigated the interaction of cationic antimicrobial peptides with the streptococcal ExPortal to provide insights into how this secretory organelle is organized and how it may be targeted by this mechanism of innate immunity.

Cationinc antimicrobial peptides: CAPs) targeted the anionic lipid microdomain of the ExPortal. Traditionally regarded as membrane permeabilizing compounds, CAPs interacted with the streptococcal membrane at concentrations that did not porate the membrane. Rather, exposure of S. pyogenes to sub-lethal CAP concentrations resulted in concomitant redistribution of anionic lipids, translocons and accessory factors to peripheral regions of the membrane. In addition, CAPs inhibited secretion of important virulence proteins, including the pathogenically relevant SpeB protease and Streptolysin O: SLO) cytolysin. One of the CAPs investigated also targets the lipid II precursor of extracellular peptidoglycan, suggesting the influence of CAPs on ExPortal organization extended beyond their affinity for negatively charged membrane lipids and that the streptococcal cell wall is involved in organizing the ExPortal.

Removal of extracellular peptidoglycan also resulted in redistribution of anionic lipids and ExPortal protein, indicating the cell wall is required for maintaining proper localization of both lipid and protein components of the ExPortal. Exposure of streptococci to lantibiotics that preferentially bind and sequester lipid II in the cytoplasmic membrane disrupted ExPortal organization and secretory function in a manner similar to CAP activity. Given that both CAPs and lantibiotics target lipid components of the cytoplasmic membrane, it appears that lipid segregation in the membrane is central to organization of the ExPortal. Establishment or maintenance of this lipid segregation could involve interaction with petidoglycan synthesis in the streptococcal membrane, as two membrane-localized synthetic enzymes, one integral: MraY), the other membrane-associated: MurN), that are required for lipid II production are localized in the same anionic lipid microdomain to where secretory translocons of the ExPortal are targeted to. This provides a potential model for the mechanism(s) organizing the ExPortal. Cytoskeletal proteins and cell wall biogenesis components which are observed to interact with cytoplasmic membrane structures to organize membrane localized processes required for cell growth and division influence the segregation of anionic lipids in the membrane, and by extension, where and how the ExPortal is organized.

Testing of this model by genetic and molecular approaches that target components of cell division and peptidoglycan will provide both further insights into how Gram-positive bacteria coordinate protein biogenesis with secretion as well as reveal novel potential targets for the development of effective therapeutics against pathogens like S. pyogenes.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7VQ30PB

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