This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Wolbachia are gram-negative bacteria carried by most insect species and filarial nematodes. Most insects harbor Wolbachia as parasites, but filarial nematodes depend on symbiotic Wolbachia infection for survival. Wolbachia's success is attributed to efficient maternal transmission and manipulations of host reproduction that favor infected females. Most of the mechanistic basis for Wolbachia-host interactions is unknown. To define host proteins interacting with Wolbachia, we focus on interaction with Wolbachia's major Surface Protein, Wsp. Based on structural homology, Wsp belongs to the pfam01617 super family whose members are surface Ag2 proteins. These proteins have conserved barrel-forming beta-strands and variable extra cellular loops. Wsp bears hallmarks of a typical antigenic protein of pathogens: highly conserved regions alternate with variable regions. This suggests that Wsp is involved in direct Wolbachia-host interactions. We have initiated affinity-chromatography approaches by bacterially expressing GST-tags fused to the entire Wsp protein or to portions containing hyper variable regions. The Wsp-GST fragments and GST alone are then bound to glutathione beads, and host extracts are run through them. Those bands enriched on the Wsp-Gst column but not on the Gst-only column are subjected to mass spectroscopy analysis. The interaction between Wsp and the proteins that have been identified by mass spectrometry will be verified by further biochemistry. Our initial studies are focusing on the interaction of the WMel Wolbachia strain and its natural host, Drosophila melanogaster. To further characterize binding specificity, we are also using Wsps from different Wolbachia strains with different hyper variable regions, including strains from the jewel wasp Nasonia and the nematode Brugia and their hosts.
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