Date of Award

2023

Degree Type

Thesis

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell & Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Matthew M. Ramsey

Abstract

Haemophilus parainfluenzae (Hp), a Gram-negative bacterium is found in different niches of the mouth, and it is very abundant in the supragingival plaque. While considered a commensal in the mouth, Hp is also a HACEK microorganism associated with endocarditis and considered an opportunistic pathogenic species. However, Hp remains a subject of limited investigation. Specifically, the genes necessary for Hp's survival and those that are conditionally essential for thriving under various environmental conditions are yet to be identified. This research addresses those questions using transposon sequencing (TnSeq) to identify the essential genome and the core essential genome in two strains of Hp (a type strain and an oral isolate). It shows that the core essential genome (essential genes conserved in others Hp strains) is composed by genes involved in genetic information processing, carbohydrate, protein, and energy metabolism. This study also uncovers aerobic and anaerobic conditionally essential genes showing that many genes essential anaerobically are also essential for aerobic biofilm growth. Analysis of transcriptomes further revealed Hp's ability to adapt to varying oxygen levels.

This study also investigates which genes and mechanisms Hp uses to evade the innate immune system allowing its survival in the bloodstream when transitioning from the mouth to other parts of the body where it can then cause diseases. Results confirm that Hp is very resistant to the killing effect of human serum and show that LPS and O-antigen aids Hp in evading the complement system and surviving in human serum. It was also observed that Hp can evade phagocytosis and stimulates the production of critical cytokines, leading to neutrophil-driven NET production. Finally, in coculture, Hp imparts opsonophagocytosis resistance to Streptococcus mitis, which is found close to Hp in the mouth. Collectively, these results indicate that Hp is resistant both to phagocytosis and the complement system and can seemingly provide or induce immune resistance in its close neighbor S. mitis.

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Available for download on Wednesday, December 04, 2024

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