A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA) has been linked to biofilm formation in B. henselae. BadA is a protein of 3036 amino acids and a member of the TAAs found in Bartonella and other Gram-negative bacteria. The function of BadA has been studied in vitro and is critical for agglutination, host cell adhesion and activation of a pro-angiogenic host response. However, very little is known about badA gene regulation or the molecular basis of biofilm formation. This work aims to determine whether BadA is necessary for the establishment of biofilms and how the bacteria regulate badA expression. Using genetic mutations, real-time cell adhesion assay, RT-qPCR, and microscopy, it was shown that BadA is required for biofilm formation. Using an in-frame complete deletion strain of badA, a reduced ability to form a biofilm was observed which was restored in the deletion strain complemented with a partial badA. Analysis of the B. henselae transcriptome shows nine highly transcribed, homologous RNAs, termed Bartonella regulatory transcript (Brt1-9). The Brts are short-sized (<200 >nucleotides), highly expressed, and located in an intergenic region indicative of small RNAs (sRNA). The Brts are predicted to form a stable stem and loop structure with a potential terminator/riboswitch region on the 3′ end. Located ~20 nucleotides downstream of each Brt is a poorly transcribed helix-turn-helix DNA binding protein gene termed transcriptional regulatory protein (trps 1-9). High brt transcription stops just before the start of the trp implicating the 3’ loop of the Brt as a terminating loop. Replacement of the trp with a gfp reporter gene shows that in the absence of the 3′ end of Brt1, gfp is transcribed. Also consistent with our findings, an increase in both the transcription of trp1 and badA and the formation of a biofilm in mutants of the brt1 gene was observed. Furthermore, to determine the role of the Trp in regulating badA, an electrophoretic mobility shift assay was carried out. The data confirms that Trp1 binds the promoter region of badA gene to regulate gene expression. In summary, the brt1/trp1 regulon affects badA transcription and biofilm formation in B. henselae. Understanding the mechanism and condition(s) by which the brt/trp regulatory system regulates badA is a plausible approach to the development of treatments that target the formation of biofilm-related diseases and persistent bacteremia in humans.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-9075 |
| Date | 02 July 2019 |
| Creators | Okaro, Udoka |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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