A genetic and functional analysis of type IV pili produced by Aeromonas bacteria
Creators: name: Barnett, Timothy Carew
Publisher: name: University Of Tasmania
Year: 2023

description: Bacteria belonging to the genus Aeromonas are ubiquitous water-borne organisms that are also present in many foods. Some strains are human gastrointestinal pathogens. However, the disease-causing mechanisms of these bacteria are not well understood. This is particularly true for intestinal colonisation, which is a critical step in the disease process. When this thesis commenced, there was evidence that filamentous surface structures (type IV pili) purified from diarrhoea-associated species had an important role in colonisation. These pili were designated \bundle-forming pili\" (Bfp) because of their tendency to form bundles linking bacteria. The initial aim of this thesis was to clone the genes encoding Bfp pili from a diarrhoeal isolate of Aeromonas veronii biovar sobria (strain BC88). Although traditional approaches (transposon mutagenesis screening of libraries with degenerate oligonucleotide probes DNA probes and a Bfp antiserum) were unsuccessful at achieving this a small region of a gene encoding the Bfp pilin protein was ultimately cloned using a degenerate PCR approach. Moreover a second type IV pilus gene cluster was also cloned from this strain. Characterisation of this newly discovered pilus family (designated Tap for \"type IV Aeromonas pilus\") was then undertaken. The tap gene cluster from A. veronii biovar sobria BC88 was shown to comprise four genes that were all transcribed in the same direction. These genes exhibited high sequence homology to genes encoded by similar gene clusters identified by other investigators from strains of A. hydrophila Vibrio cholerae and V. vulnificus. To assess the significance of Tap pili for Aeromonas virulence the distribution of the tap genes was determined in Aeromonas reference strains (including both pathogenic and non-pathogenic species) and in a range of clinical and environmental isolates of those species most commonly associated with human gastrointestinal disease. The tap cluster was present in all Aeromonas strains tested. A defined mutation in tapA from A. veronii biovar sobria BC88 was then constructed. Inactivation of this gene did not alter the ability of this strain to adhere to epithelial or intestinal cells in vitro or to colonise the intestinal tract of infant mice (colonisation and competition experiments). Furthermore the tapA mutant strain was not attenuated in its ability to cause disease in rabbits (removable intestinal tie adult rabbit diarrhoeal model; performed by Dr. M. J. Albert International Center for Diarrheal Disease Research Dhaka Bangladesh). To investigate the expression and assembly of Tap pili an antiserum was constructed against a recombinant protein produced by overexpression of tapA in Escherichia coli. Western blot analysis showed that TapA was expressed. However Tap pili on the cell surface were not seen by immune electron microscopy. (Previous studies in our laboratory have demonstrated that Bfp pill are the predominant structures expressed on the surface of Aeromonas bacteria during growth in vitro.) Hence this thesis identified a second family of type IV pili in Aeromonas species. The function and expression results obtained to date suggest that Tap pili are not as significant as Bfp pili for colonisation of the intestinal tract. However the widespread distribution of the tap genes in Aeromonas species as well as in other bacterial pathogens suggests that they are important for some aspect of the biology of these organisms."