Molecular and genetic studies on a SipD/IpaD-related virulence spike protein from immune-suppressive particles of a Drosophila parasitoid

Author

Carlo Miguel A. Sevilla, CUNY City CollegeFollow

Date of Award

2021

Document Type

Thesis

Department

Biology

First Advisor

Shubha Govind

Second Advisor

Shaneen Singh

Third Advisor

Amy Berkov

Keywords

host-parasitoid interactions, IpaD/SipD-like virulence proteins, immune suppressive extracellular vesicles, Drosophila melanogaster hemocytes, wasp egg encapsulation, innate immunity

Abstract

Parasitoid wasps are effective biological control agents that have evolved intricate host immune suppression strategies to overcome host immune defenses. The most important anti-parasite defense mechanism is encapsulation, where the host’s blood cells surround the wasp egg. In the Drosophila/Leptopilina host-parasitoid system, extracellular vesicles (EVs) are oviposited along with the wasp egg. The EVs possess spikes and contain hundreds of proteins, some of which enable wasp eggs to escape encapsulation by changing the morphology and function of hemocytes, or killing them. Previous studies have identified an abundant surface/spike protein (SSp40) from Leptopilina heterotoma EVs. While p40, a predicted membrane protein, is synthesized in the secretory cells of the venom gland apparatus, it is delivered into the host via EVs and taken up by both the macrophage and lamellocyte hemocyte types. Previous experiments suggested that p40, while still on the EV, may mediate direct EV-lamellocyte interaction and promote lamellocyte shape change and lysis. Structurally, p40 resembles the needle-tip proteins SipD and IpaD on the bacterial type 3 secretion systems (T3SS) of Gram negative bacteria Salmonella and Shigella. The bacterial T3SS delivers effector proteins into mammalian cells. To understand p40’s effects on host hemocytes, we expressed p40-RFP fusion transgenes in wild type and hopscotch^{Tumoous-lethal} (hop^Tum-l) mutant Drosophila larvae. hop^Tum-l mutant larvae have overabundant hemocytes, that form small tumors. The mutation is semi-lethal. Expression of the membrane-bound and secreted forms of p40 containing the SipD/IpaD-like domain increased viability of mutants by more than six-fold and relieved their tumor burden by more than 50%—the latter parameter being a measure of cellular immunity. Our structural analysis of the conserved p40 domain from L. heterotoma Gotheron strain from Southern France revealed only 84% sequence identity with p40 from the Lh14 strain, with 29 of the 41 amino acid substitutions being non-conservative. Homology modeling revealed that while the Lh14 and LhGoth p40 proteins show complete and precise alignment, the LhGoth protein has four additional 3₁₀ helices. These results suggest possible differences in protein folding and protein-membrane interactions in the two strains. Altogether, these observations confirm an immune-suppressive function for p40, independently of other EV proteins, possibly via direct effects on host hemocytes. They contribute to our understanding of p40’s role in parasitism by L. heterotoma and in the development of its host range.

Recommended Citation

Sevilla, Carlo Miguel A., "Molecular and genetic studies on a SipD/IpaD-related virulence spike protein from immune-suppressive particles of a Drosophila parasitoid" (2021). CUNY Academic Works.
https://academicworks.cuny.edu/cc_etds_theses/916