Date of Award

2024-05-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Katja K. Michael

Abstract

Protozoan parasites are responsible for causing numerous diseases in humans and mammals worldwide. Among them are Chagas disease (CD), caused by the parasite Trypanosoma cruzi (T. cruzi), and tegumentary leishmaniasis (TL), caused by the parasite Leishmania (Viannia). Both diseases coexist in many regions of Latin America, and current hurdles to differentially diagnose them and determine chemotherapy success need to be overcome. CD can be diagnosed by traditional serological tests and polymerase chain reaction (PCR), but disadvantages of these methods are that they may produce false negatives because the antigens used in commercial CD kits are not conserved across all T.cruzi genotypes. Additionally, to determine the treatment efficacy by conventional serology would have to rely on negative antibody seroconversion, which takes 10-20 years. On the other hand, PCR might not detect the parasite when its concentration in the blood circulation is below ~0.3-0.5 parasite equivalent/ mL of blood. For CL, traditional diagnostic methods, like microscopy, parasite culture, or PCR, have limitations due to the irregular distribution of parasites in biopsy samples. However, even when parasitemia is low, antibody levels in CD and CL patients remain high, with the CD or CL-specific anti-a-Gal antibodies being universally present. The infective form of T. cruzi expresses glycosylphosphatidylinositol (GPI)- anchored mucins (tGPI mucins) with unusual immunogenic glycans having terminal nonreducing a-Galp residues. Besides, another immunogenic moiety, b-galactofuranose (bGalf), is believed to be contained in the (GPI)-anchor. Most of those oligosaccharides are branched and their precise structures remain unknown. The CD-specific anti-a-Gal Abs show a rapid seroconversion within 2-3 years or less, following chemotherapy, making them potential candidates for diagnosis and monitoring post-chemotherapy using serological assays. Similarly, L. (V.) braziliensis expresses a family of low-molecular mass glycoinositolphospholipids (GIPLs), containing terminal a-Galp residues. So far, the exact structures of the immunodominant glycotopes responsible for eliciting IgG antibodies in CL patients have not been determined. However, neoglycoprotein NGP28b, composed of the trisaccharide Galpa1,6Galpb1,3Galfb derived from the type-2 (GIPL)-3 of Leishmania major and linked to bovine serum albumin (BSA), acts as a reliable serological BMK for L. (V.) braziliensis infection in Brazil. This suggests the existence of GIPL-3 or an analogous structure within this parasite, with its terminal trisaccharide either acting as or constituting an immunodominant glycotope. Given these findings, we investigate specific parasite-derived serological BMKs for CD and CL infection in certain New World regions utilizing chemiluminescent enzyme-linked immunosorbent assay (cELISA). Chapter 1 briefly introduces the hypothesis and specific aims of this study. Chapter 2 demonstrates the background and significance of performing this research in diagnosing and following up CD and CL. Chapter 3 describes the synthesis, optimization steps, and serological evaluation of two b-Galf-containing neoglycoproteins, NGP29b and NGP32b, believed to exist in the GPI anchor of T.cruzi using chronic CD (CCD) patient sera from endemic areas. The biological data obtained suggest that both NGPs are suitable as diagnostic biomarkers for CCD and that the branched tetrasaccharide Galfb1,3Manpa1,2[Galfb1,3]Manpa present in NGP32b is an immunodominant glycotope. In chapter 4, we studied two a-Galp-containing neoglycoproteins, NGP33b and NGP34b, that contain trisaccharide glycans believed to exist in the tGPI mucins of T. cruzi. We present their synthesis and serological evaluation using CD and CL patient sera from endemic areas. Surprisingly, the biological data indicate that the Galpα1,6 containing NGP33b is not suitable for diagnosing CD, but reacts strongly with sera of CL patients, suggesting that the Galp�1,6 moiety is an immunodominant glycotope in L.braziliensis. Also described is the partial synthesis of tetrasaccharide G38SH, another glycan structure potentially present in the T. cruzi tGPI mucins, as suggested by glycomics data. Finally, chapter 5 discusses the synthesis of a Galα1,6Galα1,3Galfβ1,3Manα-containing NGP, NGP31b, as a synthetic biomarker for L. (V.) braziliensis CL in comparison to NGP28b. Included is also a study of the glycotope size requirement. In the future, the promising new NGPs should be more deeply studied as diagnostic and/or prognostic BMKs with a larger number of patient samples. If confirmed as BMKs these NGPs could have a substantial impact on public health as they could be used for disease diagnosis and determination of treatment efficacy. One can also envision the development of an affordable lateral flow assay for point-of-care testing in developing countries where access to ELISA technologies is limited.

Language

en

Provenance

Received from ProQuest

File Size

233 p.

File Format

application/pdf

Rights Holder

Eileni Rodriguez Gil

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