Understanding the Role of Host Factors in the Modulation of Virulence Factors of Staphylococcus aureus.

Abstract

Staphylococcus aureus is adept at colonising and infecting the human host. Host factors are known to influence pathogen physiology and this thesis aimed to examine the effects of human bile and human plasmin on S. aureus.

Firstly, the effects of bile on biofilm formation in cystic fibrosis isolates of S. aureus was examined. S. aureus is one of the pathogens most frequently isolated from CF patient sputum. Bile is frequently aspirated into the lungs of these patients, often as a consequence of gastroesophageal reflux, indicating that the pathogen is likely to encounter bile in vivo. Here, certain CF isolates were shown to form a polysaccharide biofilm in the presence of bile and to produce more poly-?(1, 6)-N-acetylglucosamine in response to bile. Expression of the icaADBC operon encoding poly-?(1, 6)-N-acetylglucosamine was shown by reverse transcriptase quantitative PCR to be induced by bile indicating that bile triggers biofilm formation by upregulating icaADBC expression. Bile-induced biofilms were significantly more resistant to dispersal by oxacillin or neutrophils than biofilms grown without bile and alterations to microstructure, cell distribution and viability within a biofilm were demonstrated using confocal microscopy.

In an attempt to understand the molecular bases of differential biofilm responses to bile, the whole genome sequences of two closely related cystic fibrosis isolates from the same patient were analysed. However considerable inter-genome variation was identified and the difference in biofilm phenotype could not be explained through WGS analysis alone. Examination of a set of contemporary Irish CF clinical isolates of S. aureus uncovered a set of isolates capable of adopting either protein or polysaccharide-dependent biofilm formation. Together these findings indicate that bile is an important host signal in altering the behaviour of S. aureus and raises open questions about biofilm phenotypes relevant in vivo.

Clumping factor A (ClfA) is a staphylococcal fibrinogen-binding cell wall-anchored protein and a member of the microbial surface component recognising adhesive matrix molecules family. The most N-terminal region of the mature ClfA protein comprises three independent domains, with domains N2 and N3 adopting IgG-like folds and promoting fibrinogen binding. Nothing is known about the structure or function of domain N1. ClfA is a key virulence factor during invasive infection, promoting bacterial adhesion to fibrinogen and survival in the bloodstream. For the first time this thesis showed that processing by plasmin reduced ClfA-mediated survival in human blood and plasmin prevented ClfA-fibrinogen interactions in vitro. S. aureus activated plasmin was capable of cleaving ClfA and the cleavage site for the host endopeptidase plasmin within the N1 domain of ClfA was identified by N-terminal sequencing. Conservation of the cleavage site across seven clonal lineages of S. aureus was confirmed by amino-acid sequence alignment. These findings shed new light on the impact that proteolytic processing of ClfA has on the virulence potential of S. aureus.

In summary, this thesis demonstrates that bile is a molecular trigger facilitating S. aureus adoption of a chronic phenotype while concretely dissecting the novel dynamics of plasmin processing of cell wall-associated protein function. This research highlights the potential interplay between host interference and host exploitation by S. aureus during infection and offers a promising abundance of new research directions.