Doctoral Dissertations

Date of Award

5-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Girish Neelakanta

Committee Members

John Schaefer, Chika.C.Okafor, Oudessa Kerro Dego

Abstract

Soft ticks of the Ornithodoros genus, particularly O. turicata, are key vectors of the human relapsing fever agent, a neglected bacterial disease of public health significance. This dissertation explores two critical aspects of O. turicata biology: the role of Subolesin in innate immunity and blood feeding, and the effects of the salivary protein lipocalin on mammalian cells. Techniques like RNA extractions, quantitative real-time PCR (qRT-PCR), in vitro blood feeding, Immunoblotting analysis, RNAi-mediated silencing, Bright/Fluorescent microscopy analysis, protein array and TUNEL analysis were used in these projects.

In the first study, subolesin, a conserved protein and potential anti-tick vaccine candidate, was examined in O. turicata americanus. Several immune genes, including Toll, Lysozyme precursor (Lp), Fibrinogen-domain containing protein (FDP), Cystatin, and ML-domain containing protein (MLD), were identified and cloned. qRT-PCR revealed their developmental and tissue-specific expression. Notably, FDP expression increased significantly during blood feeding. RNAi-mediated knockdown of Subolesin reduced feeding efficiency and downregulated key immune genes, including Toll and FDP. These findings underscore subolesin's multifaceted role in tick biology and its potential as a vaccine candidate.

In the second study, the salivary protein lipocalin (Otlip) and its role in host cellular responses was examined. Cytokine array and cell culture experiments with purified Otlip and fed tick lysates showed increased expression of Insulin Growth Factor Binding Protein-3 (IGFBP-3), a pro-apoptotic protein in both murine and human cells lines. qRT-PCR and immunoblotting analysis validated the array results. Immunoblotting analysis revealed increased levels of IGFBP-3 and Caspase-3 and reduced levels of anti-apoptotic Bcl-2 upon treatment of macrophages and HaCaT cells with Otlip, suggesting cell-death induction. Cell viability assays (MTT and live/dead) confirmed higher cell death rates in GST-Otlip-treated groups compared to controls. Additionally silencing of igfbp3 expression increased the Bcl2 protein levels. Furthermore, TUNEL staining indicated apoptotic cell death in Otlip-treated macrophages. Taken together, these studies provide significant insights into the molecular mechanisms underpinning O. turicata americanus biology and highlighting its molecular players as potential for therapeutic interventions and tick control strategies.

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