Dissertations, Theses, and Capstone Projects

Date of Degree

6-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Shubha Govind

Committee Members

Mark Pezzano

Linda Spatz

Diana Bratu

Eric Lai

Subject Categories

Biology

Keywords

Notch, ROS, Immune activation and suppression, Leptopilina boulardi, Leptopilina Heterotoma, Virus-like Particles, p40, SipD/IpaD

Abstract

Drosophila melanogaster has served as an excellent model organism to study the molecular processes of innate immunity. Flies essentially lack adaptive immunity and the innate immune system is often divided into the humoral and cellular responses (Lemaitre and Hoffmann 2007). The humoral arm involves the production of antimicrobial peptides, secreted from the fat body, to combat bacterial and fungal infections. The cellular response involves the production of hemocytes (blood cells: crystal cells, plasmatocytes, and lamellocytes) in the larval lymph gland, in the sessile pools, and in circulation (Gold and Bruckner 2014). Microbial pathogens are phagocytosed by plasmatocytes whereas larger parasites such as parasitic wasp eggs are neutralized by egg encapsulation, principally by lamellocytes. The innate immune response is vital for survival against the abundant pathogens and parasites in their natural habitats. The range of microbial as well as Hymenoptera species that attack their Diptera hosts is vast. These pathogens and parasitoids have evolved strategies to either evade or suppress host immune responses (Keebaugh 2013).

This thesis contains two chapters. In Chapter 1, we focused on the mechanisms underlying host defense in response to specialist wasps of D. melanogaster, Leptopilina boulardi. Chapter 1 is already published (Small et al. 2014) and I shared first authorship with Dr. Small.

Previous experiments demonstrated that Notch (N) signaling is essential for crystal cell specification and differentiation (Duvic et al. 2002; Lebestky et al. 2003), and also promotes lamellocyte differentiation (Duvic et al. 2002). The N ligand, Serrate is expressed in the posterior signaling center (PSC), a non-hematopoietic cell population, also called the niche. Through direct contact, the PSC activates N signaling in the developing hematopoietic cells and instructs them to become crystal cells (Lebestky et al. 2003). L. boulardi infection promotes lamellocyte but inhibits crystal cell differentiation (Krzemien et al. 2010). ROS production is also activated in the PSC upon wasp infection (Sinenko et al. 2011). In Chapter 1, we demonstrate a second function for N signaling: L. boulardi parasitization inactivates N signaling in the developing lymph gland lobes; reduction of N signaling correlates with lamellocyte differentiation. We also demonstrate an unexpected link between N signaling and ROS in restricting differentiation of hematopoietic progenitors (Small et al. 2014).

In chapter 2, we focused on strategies that the generalist parasitic wasp L. heterotoma employs to actively suppress the hosts’ immune responses. Building on previous work that showed that L. boulardi infection activates NF-κB signaling in the PSC (Gueguen et al. 2013), we examined changes in the PSC and hematopoietic progenitors after L. heterotoma infection and found reduction in gene expression in the PSC, presence of VLPs around (but not within) PSC cells, and significant reduction in the progenitor population. Consistent with previous results (Chiu 2002), this reduction correlates with Caspase activation in plasmatocytes and lysis of lamellocytes, within the lymph gland and circulating hemocyte populations. These responses are mediated by virus-like particles (VLPs) produced in the L. heterotoma venom.

L. heterotoma VLPs have 4-8 spikes and the spike-to-spike distance is roughly 300 nm (Rizki and Rizki 1990). A mouse polyclonal antibody against VLPs was generated previously in our lab and immuno-electron microscopy (EM) experiments localized this protein’s origin to secretory cells of the venom gland (Chiu et al. 2006). The p40 protein is also present in large amounts in the lumen of the venom gland where VLPs undergo biogenesis and assembly (Morales et al. 2005)(Chiu et al. 2006). VLPs are ultimately deposited into the host hemocoel during the egg laying process (Chiu et al. 2006). Immuno-EM of purified mature VLPs localizes p40 to the VLP spike surface and spike termini. p40 is also present in plasmatocytes and lamellocytes of host cells. Proteomic analyses of L. heterotoma VLPs reveal more than 150 proteins, some of which are not expressed in L. boulardi (Govind lab, unpub. results).

In Chapter 2, we show (1) differential effects of L. boulardi (lamellocyte differentiation, activation of gene expression in the PSC) and L. heterotoma (cell death, repression of gene expression in PSC) on lymph gland homeostasis; (2) the subcellular localization of p40 (punctate and vesicular in plasmatocytes, nuclear in lamellocytes); (3) Rab5-dependent entry into plasmatocytes but not in lamellocytes; (4) an immune function for the PSC. Molecular characterization of p40 revealed a protein with signal sequence, a central helical domain, and C-terminal transmembrane domain. The central helical domain share structural similarity with proteins of the SipD/IpaD family, normally present on tips of Gram negative bacterial type three secretion system needles. Incubation of bacterial extracts with live lamellocytes resulted in alteration in cell morphology. We hypothesize a direct role for p40 in mediating VLP entry into lamellocytes. These studies constitute the first detailed investigation of any VLP protein and begin to uncover mechanisms of active immune suppression by VLPs. They also contribute to our understanding of the biotic nature of VLPs.

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