Characterisation of Bacterial Biofilm Formation

Abstract

Campylobacter jejuni is the largest contributor to bacterial gastroenteritis in the developed world. In addition to intestinal infections, this organism is implicated in the autoimmune neurodegenerative condition known as Guillain–Barre syndrome. C. jejuni is a common commensal organism of warm-blooded animals and avians that is passed to humans through contaminated food products or water. Biofilms are a specialised method of growth seen in many microbes and provide a means for bacteria to remain viable in harsh environmental conditions. The biofilms of C. jejuni have been hypothesised to play an important role in the transmission of infection. Herein, we aimed to investigate factors involved in C. jejuni biofilm formation as well as detailing the development of novel applications to established techniques. The first chapter of this thesis aimed to evaluate the current understanding of biofilm formation in C. jejuni. We explored factors which play important roles in biofilm formation such as chemotaxis, glycosylation, microbial metabolism, quorum sensing and stress response regulators. These elements revealed that processes which are involved in the biofilm formation of C. jejuni are frequently at odds with those required for virulence and pathogenicity. This is contrary to biofilms of other organisms such a Pseudomonas aeruginosa which require biofilms for infection. We propose that biofilms of C. jejuni play a more important role in the transmission of infection rather than pathogenesis. We then present a novel improvement to an existing method for quantification of biofilm formation. Currently, methods for quantification of biofilm formation rely on the dissolution of stained biofilm formed in microtiter plates. Solubilisation of the stain provides spectroscopic data which allows for comparisons between the relative levels of biofilm formed. The solvents used in current assays are ineffective at solubilising formed biofilm and give inconsistent results. This second chapter details the development of an alternate solvent which was able to provide complete dissolution of stained biofilm and, thus, improved the accuracy and robustness of plate-based biofilm assays. This new solvent was effective against a range of bacterial species and provides a marked improvement to microtiter plate-based methods for assessing biofilm formation. The third chapter details a combinatorial approach to expression studies in C. jejuni biofilms focusing on the role of chemotaxis signal transduction. RNA sequencing was used to examine relative differences in transcript levels between planktonic C. jejuni and those contained in a biofilm. This was combined with proteomic data obtained using iTRAQ to provide a complete profile of differential gene expression. Differential expression was observed in approximately 600 genes, with 300 displaying upregulation during biofilm formation (e.g. iron metabolism, glycan production and cell division) and 300 showing downregulation (such as metabolism and amino acid metabolism). Large tracts of the chemotaxis pathway displayed downregulation, which we investigated further using mutant C. jejuni strains deficient in the key chemotactic proteins CheV and CheW. Both of these mutant strains exhibited a significant increase in the ability to form biofilm through increased establishment of microcolonies. The absence of these signal transduction proteins also had a marked effect on motility and autoagglutination. We hypothesized that the diminished motility of the mutant strains allows for an increase in autoagglutination and, thus, more efficient microcolony formation which presents a potential pathway for modulating biofilm formation in C. jejuni. The next chapter presents a role for N-linked protein glycosylation in C. jejuni biofilm formation. Phenotypic analysis of mutant strains deficient in key enzymes, PglB and PglF, shows that in the absence of protein glycosylation, there is a substantial increase in the ability for C. jejuni to form biofilms. This appeared to be a result of increased autoagglutination leading to an increase in microcolony formation, a mechanism similar to that observed in CheV and CheW. The architecture of the formed biofilms also appears to have been altered; SEM analysis of biofilms shows a filamentous appearance in mutant strains. This may present a further means for regulating biofilm formation in C. jejuni. The work contained in this thesis identified a number of important factors which play a role in the biofilm formation of C. jejuni. It also demonstrates that coupling transcriptomic and proteomic analysis can provide a comprehensive profile of differential gene expression during biofilm formation. Many of the approaches and techniques detailed are also novel applications which have seldom been applied to the field of biofilms. These insights aim to improve not only the understanding of bacterial biofilm formation as a whole, but also the methods in which we can study them.