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A genetic and functional analysis of type IV pili produced by Aeromonas bacteria


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Barnett, Timothy Carew 1999 , 'A genetic and functional analysis of type IV pili produced by Aeromonas bacteria', PhD thesis, University of Tasmania.

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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

Item Type: Thesis - PhD
Authors/Creators:Barnett, Timothy Carew
Keywords: Aeromonas
Copyright Holders: The Author
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Copyright 1999 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 2000. Includes bibliographical references

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