Investigating the role of the V. cholerae integron/gene cassette system in biofilm formation and resistance to protozoa
- Publication Type:
- Thesis
- Issue Date:
- 2022
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Vibrio cholerae is a marine aquatic bacterium and the causal agent of the devastating diarrhoeal cholera disease that sickens millions of people each year. Persistence in the environment of this pathogen causes periodical disease outbreaks. Biofilm formation by V. cholerae on abiotic or biotic surfaces including chitin surface act as a major reservoir in the environment that also induces the natural competency of this bacterium and allows the lateral gene transfer (LGT) through uptake of exogenous DNA. Chromosome 2 of V. cholerae carries an adaptive genetic element called the integron that contains more than 150 gene cassettes of which 80-90 % are of unknown function. Integration, deletion or rearrangement of gene cassettes is dependent on a recombinase called the integron-integrase (intIA) that is induced by the bacterial SOS response, which is activated by the presence of single-stranded DNA (ssDNA) due to stalled DNA replication from damage or from acquisition of ssDNA from LGT processes.
In order to study chitin induced transformation and intIA transcription in V. cholerae, this thesis described the construction of circular and linear gene cassettes and investigated their transfer into the V. cholerae integron and demonstrating that the integron is a novel site for adding DNA for complementation of mutations in V. cholerae. Additionally, insertion of the artificial gene cassettes into attI and two different attC sites were shown to affect bacterial surface properties and biofilm formation. Finally, one of the artificial gene cassettes was used to investigate cassette transfer dynamics in V. cholerae in the presence of two bacteriovorist protozoa, the ciliate Tetrahymena pyriformis and the amoeba Acanthamoeba castellanii. This thesis shows that following internalization and packaging of V. cholerae into the food vacuoles of both protozoa, intracellular ROS induces the SOS response leading to enhanced integron-integrase expression and gene cassette recombination. In addition, this thesis shows that due to the indiscriminate feeding behaviour of protozoa, co-localisation of different species such as V. cholerae and E. coli in the same phagosome can facilitate LGT. It is shown that V. cholerae utilizes its T6SS to kill and release artificial gene cassette DNA from E. coli to make it accessible for uptake and subsequent integron-integrase mediated integration.
Taken together, this thesis, through the creation of artificial gene cassettes to study integron integration dynamics, highlights the importance of integron-associated gene cassettes in biofilm formation and shows the importance of protozoa in driving LGT-driven adaptation and evolution in V. cholerae.
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