Thursday, April 25, 2024
4/25/2024
PROJECT SUMMARY Filarial nematodes of the family Onchocercidae cause debilitating human diseases, such as lymphatic filariasis. As approximately 150 million individuals are currently infected with these nematodes, obtaining in- depth knowledge of pathogen biology will serve to address a global health issue. It is known that the filarial nematode, Brugia malayi, harbors an intracellular endosymbiotic bacterium of the Wolbachia genus, and this relationship is essential; clearance of Wolbachia from the nematode with antibiotics leads to eventual nematode death. Understanding the mechanisms by which Wolbachia maintains its intracellular survival within nematodes would therefore likely provide an important avenue towards controlling pathogenic nematode populations, but both Brugia and Wolbachia are not amenable to genetic manipulations. Discoveries of important bacterium:nematode interactions at the molecular level, therefore, have proven exceedingly difficult. In this proposal, our goals are to utilize proteins from Wolbachia to genetically and biochemically dissect conserved pathways of endolysosomal membrane dynamics in yeast. These secreted “effector” proteins are known to alter host processes in order to support the survival of the bacterium in the eukaryotic host and to ensure its own reproduction, and are therefore potent reagents that impact eukaryotic physiology. To this end, my laboratory has employed the budding yeast, Saccharomoyces cerevisiae (Sce), as a model system towards the discovery of bacterial proteins that modulate eukaryotic cellular biology, with a focus on those proteins which inhibit intracellular membrane fusion and protein trafficking pathways. In a previous screen of candidate wBm secreted effector proteins, we have already identified proteins from wBm that have the ability to manipulate eukaryotic biology. In this work, we show that one such protein, wBm0152, strongly inhibits endosome:vacuole trafficking pathways in vitro. This inhibition appears to result from modulation of the conserved ESCRT complex. As wBm is known to alter membrane dynamics in its host during its symbiosis, and coupled with the fact that regulation of membrane dynamics is strongly conserved throughout eukaryotes, the detailed genetic, molecular, and biochemical studies carried out in this proposal will be applicable to wBm:B. malayi interactions, and thus, human filarial diseases. Finally, leveraging our laboratory's strengths in microbiology, cellular biology, and protein/ lipid biochemistry, we will carefully detail the biochemical activity of this Wolbachia-derived ESCRT modulator and identify important regulators and binding partners in yeast, which are likely conserved in Brugia. This work will begin to describe heretofore unknown wBm:B. malayi interactions, thus providing novel insight into not only Brugia physiology, but also provide new insight into ESCRT-dependent activities in eukaryotes.
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