Characterisation of the FitAB Toxin-Antitoxin System in Neisseria gonorrhoeae
Warrender, A. (2019). Characterisation of the FitAB Toxin-Antitoxin System in Neisseria gonorrhoeae (Thesis, Master of Science (Research) (MSc(Research))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12608
Permanent Research Commons link: https://hdl.handle.net/10289/12608
Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. In a standard infection, N. gonorrhoeae invade epithelial cells lining the urogenital tract and exit into the sub-epithelial layer. This triggers an immune response that causes the noticeable symptoms of gonorrhoea. Asymptomatic infection occurs when the bacteria persist inside epithelial cells, avoiding detection by the human immune system. Bacterial persistence has been linked to the development of antibiotic resistance, and as such, is a major contributing factor to the success of the pathogen. Mechanisms of adherence and invasion by N. gonorrhoeae have been well researched, but little is known about the intracellular lifecycle of the bacteria during infection and persistence. Toxin-antitoxin (TA) systems have been linked to the formation of persistent populations in bacteria however the role of TA systems in N. gonorrhoeae is yet to be investigated. In this thesis, the role of a VapBC TA system in intracellular replication of N. gonorrhoeae is investigated and activity of the toxic VapC protein is characterised. The TA system, FitAB, is a protein-protein complex comprised of an antitoxin, FitA, and a toxin, FitB. Activity of FitB is inhibited when in complex with FitA, and the complex is auto-regulatory. Biochemical activity screens using RNA pentaprobe substrates to characterise the activity of FitB show FitB cleaves single-stranded RNA pentaprobes. However, FitB does not cleave short RNA oligonucleotides of the same sequence(s). These results indicate FitB is a magnesium-dependent sequence-specific ribonuclease that cleaves RNA based on sequence and secondary structure. The potential role of FitAB in persistence of N. gonorrhoeae was investigated using intracellular replication experiments. Intracellular replication of a N. gonorrhoeae fitAB gene deletion strain showed the most pronounced phenotype seen for a single TA deletion. Intracellular replication of the fitAB gene deletion strain is considerably faster than the wild-type within epithelial cells. However, a sudden cell death event occurs in the gene deletion strain suggesting the TA system is essential for sustainable, long-term growth during infection. An understanding of how FitAB regulates intracellular growth will provide insight into the intracellular lifecycle of N. gonorrhoeae and the formation of persistent populations.
The University of Waikato
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