Date of Award

2020-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Igor C. Almeida

Abstract

Chagas disease (ChD) is a neglected tropical disease (NTD) caused by the protozoan parasite, Trypanosoma cruzi. It is transmitted by the insect-vector triatomine (popular known as kissing bug), blood transfusion, organ transplantation, congenitally, and contaminated foods and juices. T. cruzi has evolved several strategies to invade the host cells, including the release extracellular vesicles (EVs), which assist pathogen survival and its replication within the host. T. cruzi is covered with highly glycosylated surface molecules such as glycoproteins and glycolipids, which are shown to be involved in the interaction with host immune cells. These molecules are highly immunogenic and reactive with chronic patients’ sera, eliciting a strong anti-parasitic immune response, which includes the production of the highly abundant, immunodominant, and protective anti-α-Gal antibodies, which are critical for controlling the parasitemia in the chronic phase of ChD. α-Gal-Containing glycoconjugates are also released through EVs from the parasite, indicating therefore that these vesicles might play an important role in persistence of T. cruzi in the mammalian host and evasion from host immunity. Our first hypothesis is that T. cruzi virulence factors released in extracellular vesicles (EVs) are highly antigenic and reactive with sera from chronic Chagas disease (ChD) patients and could be used as biomarkers for reliable diagnosis of ChD. Our second hypothesis is that major T. cruzi virulence factors (e.g., proteins and glycoproteins) and bioactive molecules (e.g., lipids) are differentially secreted in EVs from major parasite genotypes with distinct virulence and pathogenic traits (Specific Aim 1). Our third hypothesis is that T. cruzi EVs from major parasite genotypes could differentially modulate host-cell infection rate in vitro in a dose- and time-dependent manner (Specific Aim 2). In order to address the first and second hypotheses, TCT-Secr, α-Gal-enriched EVs (α-Gal(+) TCT-EVs) and α-Gal-depleted EVs (α-Gal(-) TCT-EVs) present in the TCT-Secr, from different parasite genotypes and strains, will be tested against ChD human serum pool (ChHSP), normal human serum pool (NHSP), and anti α-Gal Abs for the reactivity against these sera. We will then perform proteomic and lipidomic analysis of TCT-Secr, α-Gal(+) TCT-EVs and α-Gal(-) TCT-EVs from different parasite genotypes and strains to study the protein and lipid composition of these fractions (Specific Aim 1). Thereafter, to address the third hypothesis, we will measure the T. cruzi infection in vitro (% of infected cells, and number of the parasites per infected cells) with different parasite genotypes and strains, after pre-treatment of the cells with TCT-Secr and TCT-EVs (Specific Aim 2). Here, we have observed that TCT-Secr and TCT-EVs derived from T. cruzi infective forms from three major genotypes are highly enriched with α-Gal epitopes, and are highly reactive with chronic ChD. We have been able to successfully purify and enrich these epitopes (α-Gal(+) TCT-EVs) by performing immunoaffinity chromatography (IAC) utilizing a lectin which specifically binds to α-Gal epitopes. These purified α-Gal(+) fractions showed high reactivity with Ch anti α-Gal Ab in comparison with the fractions (α-Gal(-)) depleted of the α-Gal epitopes. The proteomic analysis of the TCT-Secr and TCT-EVs has shown that the parasites release various T. cruzi surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins such as mucins of the TcMUC II family, trans-sialidases (TS), mucin-associated surface proteins (MASP), GP63, and other glycoproteins that are involved in invasion of host cells and evasion from the host immune system. The proteomic analysis of α-Gal(+) TCT-EVs showed that TcMUCII mucins are a major component of those EVs. TcMUCII mucins have been previously shown to be α-galactosylated. The α-Gal(-) TCT-EVs, on the other hand, were enriched with the glycoproteins previously known to contain little or no α-Gal epitopes such as TS, MASP, and GP63 families. Furthermore, we have found various lipid species involved in the parasite’s virulence in the TCT-Secr and TCT-EVs, such as plasmalogens (various ether-phosphatidylcholine (PCe) and ether-phosphatidylethanolamine (PEe) species), as well as lyso-phosphatidylcholine (LPC). Plasmalogens are known to be involved in membrane bending and EV fusion, and LPCs are known to have platelet-activating factor (PAF)-like activity, which could be involved in pathophysiology of ChD. Finally, we have observed a significant increase in host-cell infection rate as well in the number of parasites per cell after pre-treatment with TCT-Secr. Taken together, our results strongly indicate that the TCT-EVs could function as a mechanism of immune evasion, by capturing the highly abundant anti-α-Gal antibodies, and as virulence factor, by making the cells more susceptible to infection.

Language

en

Provenance

Received from ProQuest

File Size

251 pages

File Format

application/pdf

Rights Holder

Nasim Karimi Hosseini

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