DOI

10.17077/etd.mcvgr7mo

Document Type

Dissertation

Date of Degree

Spring 2017

Access Restrictions

Access restricted until 07/13/2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Wilson, Mary E.

First Committee Member

Houtman, Jon

Second Committee Member

Maury, Wendy

Third Committee Member

Zabner, Joseph

Fourth Committee Member

Yahr, Timothy

Abstract

The Leishmania spp. are kinetoplastid protozoan parasites that cause a spectrum of highly prevalent and neglected tropical diseases known as leishmaniasis. The parasites must undergo two life forms during their life cycle: the extracellular promastigote life stage within the sand fly vector, and the intracellular amastigote life stage after internalization of host phagocytic cells. In the extracellular life stage, Leishmania promastigotes reside and develop to their infectious metacyclic form solely in the gut lumen of the sand fly, a process known as metacyclogenesis. During this process, other organisms that co-inhabit the sand fly gut, collectively known as the microbiome, influence parasite development. Based on the hypothesis that vector gut microbiota influence the development of parasite virulence, we sequenced midgut microbiomes of the sand fly Lutzomyia longipalpis with or without L. infantum infection. Sucrose fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in bacterial richness, which eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Furthermore, parasites altered the relative abundance of several bacterial phylogenies, including Pseudomonas and Serratia. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and consequent development to infectious metacyclic forms, and revealing the level of microbial diversity may induce flies resistant to infection. Together, these data suggest the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation prior to disease transmission.

During the intracellular amastigote life form, macrophages are the primary cell type to phagocytize parasites. The effect of secreted factors such as exosomes from Leishmania-infected human cells and their effect on the immune response has not been extensively investigated. In this thesis, we characterized the proteome of primary human donor monocyte-derived macrophage (MDM) exosomes during L. infantum infection compared to donor-matched uninfected controls, and determined their impact on naïve MDMs measured by cytokine gene expression and resistance to subsequent parasite infection. Proteomic comparisons of infected and uninfected MDM exosomes were made using stable isotopic dimethyl labeling LC-MS/MS technology. A total of 484 human proteins were identified between four donors. Proteins significantly less abundant in exosomes derived from infected MDMs were matrix metalloprotease 9, galectin-3 binding protein, and several Annexins and histone proteins. Proteins more abundant included galectin-1, galectin-9, and serotransferrin and transferrin receptor 1. Interestingly, class I and class II MHC protein chains were differentially abundant in our samples. Furthermore, we observed several Leishmania spp. proteins in exosomes from infected MDMs as well. Naïve MDMs pretreated with exosomes from infected or uninfected MDM for 4 hours were not more resistant to L. infantum infection nor displayed increased gene expression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 or TNF-α. To date, the work presented in this thesis is the first to comprehensively identify the proteome in primary human MDM exosomes during Leishmania spp. infection, and to determine the impact of these exosomes on the immune response of other naïve human MDMs.

Public Abstract

Leishmaniasis is caused by parasites that infect humans and animals that live in tropical climates. Parasites have two phases to their life cycle; 1) exclusively inside the gut of a sand fly during the extracellular life stage, and 2) after a sand fly transmits parasites to invade cells in the body and cause disease in the intracellular life stage. My research focuses on both the extra- and intracellular life stages of Leishmania, the protozoan parasite and causative agent of leishmaniasis.

During the extracellular life stage in the sand fly gut, parasites must replicate and differentiate to their infective form while interacting with other microorganisms such as bacteria that co-inhabit the gut. We found parasites alter the composition and phylogenetic diversity of the microbes in the gut of the sand fly during infection. However, after disruption of the gut bacteria via antibiotics, parasites die and are unable to be transmitted to mammalian hosts. This suggests parasites require the presence of bacteria in the gut of the sand fly to survive and complete their life cycle.

In the case of a successful transmission to mammals via the sand fly bite, parasites are internalized by white blood cells (such as macrophages) where they transform to their intracellular life form. During this stage, parasites disrupt the cross-talk of secreted factors and gene expression between host cells and subsequently suppress the immune response. We found the proteins present in the secreted nanovesicles from infected human macrophages are altered during infection, and also include parasite proteins, which impact the immune response. In the future we would like to further investigate how the interactions between parasites and their microenvironments affect pathogenesis of infection.

Keywords

Exosomes, Leishmania, Macrophage, Microbiome, Parasite, Sand Fly

Pages

xix, 202 pages

Bibliography

Includes bibliographical references (pages 186-202).

Copyright

Copyright © 2017 Patrick Hogan Kelly

Included in

Microbiology Commons

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