Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences - Microbiology & Immunology

Committee Director

Girish Neelakanta

Committee Member

Hameeda Sultana

Committee Member

Piotr Kraj

Committee Member

John D. Catravas

Abstract

Vector-borne diseases (VBDs) are illnesses transmitted to humans and other animals by arthropods such as ticks, mosquitoes, and fleas. These arthropod vectors transmit infectious pathogens such as viruses, bacteria, and protozoa, to humans during blood-feeding. We have very few control strategies to treat or control these diseases. Human anaplasmosis, caused by the bacterium Anaplasma phagocytophilum, is the second most common tick-borne disease in the United States. This work defines three studies elucidating Anaplasma phagocytophilum-mediated modulation of cell signaling in mammalian cells and arthropod vector Ixodes scapularis ticks.

The first study focused on mammalian PI3 kinases signaling in regulating cell cycle gene expression during A. phagocytophilum infection. Using the human megakaryocytic cell line, MEG-01, we observed a differential expression of cell cycle genes in these cells upon A. phagocytophilum infection. Both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and IκB were higher at early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in a significant reduction in the bacterial load and the expression of cell cycle gene expression. These results suggest a role for PI3K-Akt-NF-κB signaling in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

The second study showed that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of the arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 significantly affected iafgp gene expression and bacterial burden in ticks during acquisition from the murine host. The electrophoretic mobility shift assays (EMSAs) revealed that both the bacterium and AP-1 protein influence iafgp promoter and expression. The luciferase assays demonstrated that a 700 bp upstream region of the antifreeze gene is sufficient for AP-1 binding to drive iafgp gene expression. Furthermore, survival assays revealed that ap-1 deficient ticks were more susceptible to cold than the mock control ticks. These data show that AP-1 acts as an upstream transcriptional activator to drive the iafgp expression that is critical for A. phagocytophilum survival in I. scapularis ticks.

The third study identified and characterized the circadian components in I. scapularis. The identification of the core clock genes in ticks was made using bioinformatic analysis from the Ixodes scapularis genome. Core clock genes like clock1 and bmal1 were upregulated upon tick feeding on the murine host. RNAi-mediated knockdown of the arthropod clock1 gene resulted in an increased bacterial transmission from ticks to the murine host. These results indicate that arthropod clock-mediated signaling is essential for transmitting A. phagocytophilum from tick to the vertebrate host. Taken together, these studies highlight several undefined mechanisms that A. phagocytophilum modulates for its survival in mammalian cells and ticks.

DOI

10.25777/rh8x-r641

ISBN

9798516059384

ORCID

0000-0002-8483-9940

Available for download on Friday, June 10, 2022

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