Sharp, Lindsay Joanne; (2003) The study of exported proteins from bacteria causing local inflammation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Procedures taken to identify exported virulence factors from bacteria causing inflammatory diseases have been described. Porphyromonas gingivalis Lipid A-associated proteins (LAPs) were extracted and shown to induce IL-6 production by human cells. The cytokine-inducing LAP was isolated and shown to be homologous to a conserved domain present in the P. gingivalis adhesin/haemagglutinin family of proteins including the gingipains, HagA and Tla. It was demonstrated that arg-gingipain is able to degrade IL-1β and eradicate its ability to induce IL-6. Thus it appears that domains within the same molecule have opposing effects on cytokine levels and may therefore contribute to the chronic inflammatory disease periodontitis. To identify exported proteins from Staphylococcus aureus, antisera raised against surface-associated material from this bacterium were used to screen a S. aureus genomic DNA library. Proteins expressed by the clones recognised by the antibody were identified. These proteins include protein A, SdrE and a novel serine rich glycoprotein (Sta). Although the exact function of Sta was not elucidated, it is a member of the MSCRAMM family of proteins. The development of a functional genomic assay to identify genes encoding cytokine-modulating proteins, using an Escherichia coli periplasmic expression system, was investigated. This involves testing periplasmic-proteins (PPs) from bacterial genomic libraries, for cytokine inducing activity. A problem with this approach was contamination of PPs with lipopolysaccharide. A mutant E. coli strain defective in lipopolysaccharide biosynthesis was investigated for this purpose. It was shown that PPs from this strain induced cytokine production in the presence of anti-CD14 antibody but not in the presence of polymyxin B suggesting that this activity could be assigned to a polymyxin B-binding protein and not LPS. Affinity purification of the PPs showed that; peptidylprolyl-cis-trans-isomerase and flagellin bound to polymyxin B but these pure proteins did not induce cytokine production. The cytokine inducing PP remains unidentified.

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