Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Jason Carlyon

Second Advisor

Cynthia Cornelissen

Third Advisor

Richard Marconi

Fourth Advisor

John Ryan

Abstract

Granulocytic anaplasmosis, caused by the obligate intracellular bacterium, Anaplasma phagocytophilum, is the second most common tick-transmitted disease in the United States. As an obligate intracellular pathogen, it must enter a host cell to survive, replicate and cause disease, therefore, delineating the mechanisms and microbial proteins responsible for the initial binding and entry step could identify novel therapeutic targets and preventative measures against this disease. Previous work identified the binding domains for three A. phagocytophilum adhesins, OmpA59-74, AipA9-21, and Asp14113-124. Herein, we demonstrate that C57BL/6J mice immunized with a cocktail of KLH- conjugated peptides corresponding to the binding domains followed by challenge with A. phagocytophilum elicited antibodies against AipA9-21 and Asp14113-124 and activated IFN-g producing CD8+ T cells. This resulted in a pronounced reduction in bacterial load in immunized mice compared to controls. A follow up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to elicit antibodies that inhibit A. phagocytophilum cellular infection and reduce the peripheral bacterial blood load. While load was reduced in vivo, infection persisted suggesting the contributions of additional adhesins. AipB was identified as a potential invasin and its binding domain was narrowed down to amino acids 80 to 100. CD13 and CD18 were identified as the putative binding partners for AipA and AipB, respectively, and their relevance for establishing A. phagocytophilum infection was confirmed. Collectively these data indicate the relevance of A. phagocytophilum adhesins to establishing infection in vitro and in vivo, while shedding light on multiple novel host cell receptors utilized by an obligate intracellular pathogen for binding and entry. Like other intracellular pathogens, A. phagocytophilum is highly dependent on cholesterol acquisition for growth. The bacteria poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine, which functionally inhibits ASM. Herein we demonstrated that ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner, and A. phagocytophilum is capable of co-opting ASM-independent lipid sources. Collectively these data not only provide a basic understanding of how this bacteria establishes infection filling in previous knowledge gaps, but they work cooperatively to elucidate several targets that can be used in new therapeutic and preventative measures against this potentially debilitating disease. These studies also highlight the importance of potentially utilizing desipramine as an alternative drug in treating A. phagocytophilum infection.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

7-9-2021

Available for download on Tuesday, May 22, 2221

Included in

Bacteria Commons

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