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Inhibition of autophagy by Entercoccus faecalis

Nathan Shankar Ph.D.

Principal Investgator

Agency: Presbyterian Health Foundation Seed Grant

Abstract

Phagosome trafficking and maturation is an important process that governs elimination of phagocytized pathogens and culminates with the formation of phagolysosomes which effect killing of its cargo. Autophagy is a highly conserved pathway designed to maintain eukaryotic cellular homeostasis by degrading dysfunctional proteins and damaged organelles in the cytosol. Recent studies have demonstrated that autophagy also serves as an important defense mechanism to clear intracellular microorganisms. The autophagy process involves the formation of double membraned vesicles that sequester cytosolic cargo and transport them for fusion to lysosomes for degradation. Several pathogenic bacteria have evolved ways to interfere with phagosome maturation and autophagy in order to survive in host cells.

Enterococci rank among the most common nocosomial pathogens infecting the bloodstream, surgical sites and urinary tract. Altough they are known to persist for extended periods of time in host cells such as macrophages, the fate of phagosomes carrying E. faecalis, and the mechanism(s) for this persistence, have not been studied until now.

In this proposal, we present preliminary data to show that the number of phagocytized E. faecalis E99 in RAW264.7 macrophages stays constant until about 12h postinfection. Examination of the autophagic process in E. faecalis infected RAW cells revealed that internalized E99 appeared to be surrounded only by single layer membranous vesicles, and western blot analysis showed lower level of autophagy proteins LC3-II up to 24h post infection, compared with uninfected cells.

These observations implied that E. faecalis infection may inhibit autophagy in macrophages to promote bacterial survival. When autophagy was induced by pharmacologic inducer rapamycin in infected RAW cells, it could significantly increase clearance of the bacteria in vitro. Based on these preliminary studies, we propose to employ molecular and cellular biological techniques to investigate the intracellular trafficking of E. faecalis and the role of autophagy in influencing the survival of E. faecalis in macrophages. The results of these studies may offer a significant new approach to therapeutic intervention for the treatment of serious enterococcal infections.