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Investigating the impact of the FIC domain Bartonella quintana effector protein, BepA1, on human innate immune responses

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thesis
posted on 08.02.2022, 01:51 by Ramela, Aman-Deep

Bartonella are known as stealth pathogens that subvert the host immune system, using an arsenal of pathogenicity factors that enable them to survive and persist in their reservoir host. The focus of our study is B. quintana, a historically famous pathogen that caused trench fever in soldiers during World War 1, but today causes an increasing number of urban trench fever cases in vulnerable populations, such as homeless or immunocompromised groups. After exposure to B. quintana, the bacteria traverse the skin and enter the bloodstream, infecting erythrocytes. B. quintana encounter host immune cells in the vascular system, but are not killed, hijacking these cells to disseminate into the lymphatic system where they eventually make their way into the bloodstream. How B. quintana modulates host immune responses to its favour during early infection is very poorly understood due to the current lack of research surrounding this pathogen. B. quintana, like many Bartonella species, possesses a Type IV Secretion System, a large protein complex that is used to inject Bartonella Effector Proteins, Beps, directly into target host cells. B. quintana encodes six BEP proteins, of which BepA1 and BepA2 are the best studied. BepA1 contains a FIC domain, which in other pathogens have been shown to catalyse addition of an AMP moiety to target host proteins, interrupting function and downstream signalling. This process, AMPylation, is used by various bacterial pathogens to interfere with actin cytoskeletal signalling to inhibit phagocytosis. However, the AMPylation function in B. quintana BepA1 remains to be shown. BepA2 has been shown to inhibit apoptosis once translocated into endothelial cells. Previous studies in our lab indicated that BepA1 may play a role in suppression of the innate immune system. Wild type BepA1-transfected HeLa-229 cells showed decreased levels of IL-6 and IL-8 in comparison to an empty vector control. We thus hypothesised that BepA1 is being translocated into host cells to AMPylate a signalling protein/s to suppress immune signalling and immune cell recruitment, enabling the survival and persistence of B. quintana in the bloodstream. Our study thus focused on better understanding the host-pathogen interactions between B. quintana and the human innate immune response. We began by broadly characterising the innate immune responses of human cells transfected with the wild type B. quintana BepA1 protein. HeLa-229 cells were transfected with a vector expressing the BepA1 wild type protein, or a catalytically dead BepA1 mutant protein. Cells were stimulated with TNF-α, and cytokine ii and chemokine expression was evaluated. These observations were validated via qPCR analysis of the transfected, TNF-α treated cells. Lastly, we aimed to generate a targeted BepA1_BepA2 B. quintana deletion mutant to further study the consequences on host-pathogen interactions. Unfortunately, we were unable to generate this mutant. Our work provides the first evidence that we know of that suggests a bacterial effector with a FIC domain is involved in manipulation of cytokine and chemokine expression. Our work provides a foundation for a better understanding of the early stages of Bartonella infection and pathogenesis. With this research, we hope to shed light on how B. quintana hampers immune signalling during infection, leading to its intracellular persistence and survival in the reservoir host.

History

Copyright Date

08/02/2022

Date of Award

08/02/2022

Publisher

Victoria University of Wellington - Te Herenga Waka

Rights License

Author Retains Copyright

Degree Discipline

Microbiology

Degree Grantor

Victoria University of Wellington - Te Herenga Waka

Degree Level

Masters

Degree Name

Master of Science

ANZSRC Type Of Activity code

4 EXPERIMENTAL RESEARCH

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

MacKichan, Joanna