The regulation and biological activity of cell surface virulence determinants in model opportunist aerobic and anaerobic bacterial pathogens

Abstract

Burkholderia cepacia is an aerobic Gram- negative bacterium originally described as the cause of soft rot in onions but now recognised as a serious human pathogen most notably in patients with cystic fibrosis (CF), the most common, fatal inherited disease affecting Caucasian populations. Bacteroides fragilis, a Gram- negative commensal associated with the mucosal surface of the human colon, is the most frequently isolated anaerobic bacterium from clinical specimens and is increasingly implicated as an important source of endotoxin in gut-derived sepsis. Previously, both organisms were considered to pose little hazard to human health and consequently, their pathogenesis and virulence factors are poorly understood. The cell surface components of a bacterium are classic virulence determinants and the influence of growth environment on the plasticity of the bacterial surface is well -established. This thesis considers the environmental regulation and biological activity of putative virulence factors for both organisms.

Initial studies focused on the expression and antigenicity of the cell surface components; lipopolysaccharide (LPS), exopolysaccharide (EPS) and outer membrane from several representative strains. To determine the influence of growth environment, these components were investigated under different culture conditions and extraction methods. Results demonstrated that as with other Gram- negative organisms, the composition of outer membrane proteins of both B. cepacia and B. fragilis were influenced by cultural conditions with bacteria inducing or repressing protein structures presumably to influence overall permeability and survival. The EPS and LPS of B. fragilis also varied with environmental growth condition. Interestingly, extraction method was found to influence the LPS structure of B. cepacia including the loss of the distinctive rough LPS phenotype of an 'epidemic' strain.

B. cepacia and B. fragilis exhibited a greater biological activity than previously recognised both in terms of endotoxicity and cytokine induction. For B. cepacia the capacity to induce the proinflammatory cytokines TNF -a and IL -8 from several cell types was significantly, and unexpectedly, higher compared to the other major CF pathogen, Pseudomonas aeruginosa. This enhanced inflammatory potential of B. cepacia was not due to a more efficient LPS signalling pathway. As both CF pathogens appeared to induce TNF -a in a similar manner, the combined effect of both species was examined. Surprisingly, when P. aeruginosa was present in increasing amounts compared to B. cepacia, cytokine levels were down -regulated. These results indicate that B. cepacia has a major potential to cause immune - mediated damage and concurrent colonisation with P. aeruginosa may modulate this effect. For B. fragilis cytokine levels were compared to Escherichia coli, a facultative anaerobic commensal considered of great importance in gut- derived sepsis due to its extremely active LPS. TNF -a levels induced by B. fragilis were 20 -fold lower than E. coli. However, considering the predominance of Bacteroides species in the gut, outnumbering facultative organisms by 20 -300 fold, results imply that as a population B. fragilis may possess as much biological potential as E. coli. Thus, B. fragilis may play a vital role in gut- derived sepsis. The relevance of these findings to the understanding of B. cepacia in CF and of B. fragilis in sepsis is discussed.