Dissertations, Theses, and Capstone Projects

Date of Degree

5-2018

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Shubha Govind

Committee Members

Thomas Briese

Reza Khayat

Shaneen Singh

Jayne Raper

Subject Categories

Biochemistry | Bioinformatics | Entomology | Parasitology

Keywords

Leptopilina, venom, virus-like particles, immune suppression, parasitoids, parasitic wasps

Abstract

Parasitic wasps act as keystone species in natural ecosystems. Adept at suppressing immunity of their insect hosts, these natural enemies of insect pests are used for biocontrol in many parts of the world. Female parasitic wasps of the closely-related species Leptopilina heterotoma (Lh), a generalist of many Drosophilia flies, and Leptopilina boulardi (Lb), a specialist on flies of the melanogaster subgroup, produce venom and virus-like particles (VLPs) in their long gland-reservoir complexes, a secretory organ connected to ovipositors. Venom and VLPs are deposited, along with wasp eggs, into the body of the wasp’s larval fly host during infection. The bioactivity of VLPs is directly linked to suppression of cellular immunity in larval fly hosts and the parasitic success of the wasps. Venom and VLP proteins modulate host immunity to allow wasps to safely develop in the host’s body cavity, while feeding on the developing fly tissues.

To understand VLP biogenesis, discover infection-related bioactive products, and provide a resource for molecular investigations of the parasites of Drosophila, we analyzed transcripts of the long-gland reservoirs of Lh NY. In Chapter 1, we describe 823 unigenes of which approximately 200 were unannotated and/or novel. 75% and 25% of the remaining transcripts were similar to conserved cell physiology and putative venom-effector proteins, respectively (1). The large-scale conservation found between Lh transcripts and genes of stinging Apocrita species suggests that the findings of this thesis will be pertinent to research on honeybee, various ants, and the well-characterized ectoparasitoid jewel wasp, Nasonia vitripennis. Chapter 1 was among the first set of transcriptomic studies of a virulent parasitic wasp of D. melanogaster.

During infection, VLPs of Lh (Lh VLPs) lyse host lamellocytes, a large and sticky blood cell type that sequesters wasp eggs. Using a polyclonal antibody, the Govind lab identified a 40 kDa protein (“p40”) localized to the surface and spike tips of Lh and Lv VLPs. Because p40 is necessary for VLP-mediated lamellocyte lysis, we used proteomics to identify its sequence and those of other VLP proteins potentially critical for suppressing host immunity and contributing to Lh’s broad host range (Chapter 2). The Lh VLP proteome is surprisingly large (~160 proteins) and non-viral. ~ 40% of the proteome is enriched with proteins in a profile similar to that of eukaryotic extracellular microvesicles (Class 1). Proteins characteristic of immune modulation or the infection activities/mechanisms (Class 2) are present; some of these belong to expanded gene families. A majority of the sequences without known homologs (Class 3), including p40, appear to not be expressed in Lb.

p40 is predicted to be a transmembrane protein and its primary structure lacks similarity to known proteins. Surprisingly, it contains the SipD/IpaD protein domain from Gram-negative type III secretion systems of Shigella and Salmonella spp. Tertiary structure-based predictions indicate that, like SipD and IpaD, p40 modulates the actin-based cytoskeleton of host cells, which is suggestive of how VLPs enter non-phagocytic lamellocytes and induce the cell shape changes that precede VLP-induced lysis. Proteomic results for Lh VLPs suggest that VLPs represent a new kind of organelle, with elements of secretion systems from eukaryotes and prokaryotes. VLPs have therefore been renamed Mixed-Strategy Extracellular Vesicles, or MSEVs (2).

In Chapter 3, we examined the structure and function of a representative GTPase, a member of an abundant protein family not expressed in Lb transcriptomes. Sequence analyses of eight GTPases and their gene structures suggest a mixed prokaryotic/eukaryotic character of these proteins. SmGTPase01, when expressed in budding yeast, localized to the yeast vacuole, a homolog of the eukaryotic lysosome. An unbiased genome-wide interaction study suggested that SmGTPase01 compromises intracellular trafficking and lysosomal functions in host cells. MSEV localization experiments conducted in fly macrophages showed distorted phagolysosmal morphologies suggesting that SmGTPase01 functions in intracellular transport of venom proteins (e.g., VLPs) in host blood cells. Investigations to test these predictions are underway.

The Drosophila-Leptopilina host-parasite pair is an emerging immunity-virulence model as it easily lends itself to powerful molecular-genetic and cell biology approaches commonly used in the Drosophila field. This study provides the first comprehensive inventory of a Lh’s MSEV proteins. Their identities can form the basis of new hypotheses regarding MSEV origins and help determine phylogenetic relationships between the close and distant relatives of Leptopilina wasps. Availability of physical clones and sequences will help design approaches to test virulence protein expression and function experimentally. These studies will impact our understanding of the pivotal roles parasitic wasps play in shaping natural insect communities. The fly-wasp/host-parasite model is of increasing interest to parasitologists, immunologists, neurobiologists, evolutionary biologists, and agriculture scientists. While, the wasps are significantly less well-characterized than their Drosophila hosts (and will remain so in the near future), continued efforts to study this fascinating model promise to deliver unexpected insights with potential applications to human health and crop productivity.

Download

Share

COinS