The ability of Staphylococcus aureus to adhere to the extracellular matrix and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of orthopaedic-device related infections. S. aureus possesses many adhesion proteins on its surface, but it is not known how they interact with each other to form stable interactions with the substrate.A novel method was developed for extracting adhesins from the S. aureus cell wall, which could then be further analysed. The protocol involves using a FastPrep instrument to mechanically disrupt the cell walls resulting in native cell walls. Ionically and covalently bound proteins were then solubilised using sodium dodecyl sulphate (SDS) and lysostaphin, respectively. Western blot analysis of covalently bound proteins using anti-protein A and anticlumping factor A sera showed that S. aureus produces most surface proteins in early growth, and less in postexponential and stationary growth.Immuno-gold labelling of protein A, and clumping factor A was observed all over the bacteria and showed no distinct surface distribution pattern. However, this labelling showed expression of surface associated proteins varied in a growth-phase dependent and cell-density dependent manner.

The accessory gene regulator (agr) and staphylococcal accessory regulator (sar) loci are important regulators of toxin production in Staphylococcus aureus. In this study we examined how environmental conditions degree of aeration and salt concentration - affect the transcription and translation of mRNAs for alpha-haemolysin (Hla) and serine protease (Ssp) via these pathways and influence the stability of these proteins. Using Northern analysis, we have confirmed earlier observations that sarA is involved in the upregulation of RNAIII, the effector molecule encoded by the agr locus. However, this effect was abolished in highly aerated cultures. While sarA does appear to have an up-regulatory effect on hla transcription that is independent of agr, we propose that the PC1839 (sarA) mutant produces less alpha-haemolysin activity mainly as a result of post-translational inactivation by proteases. The most obvious phenotypic feature of PC1839 (sarA) is the upregulation of proteases. In this study we show that ssp is repressed by SarA at the transcriptional level. Western analysis using an anti-alpha-haemolysin antibody identified a major breakdown product that is only present in the supernatant of strains that are overexpressing serine protease. We have also confirmed that agr exerts a significant regulatory influence on hla at the level of translation, as well as transcription. Finally, the addition of salt upregulates ssp transcription and dramatically downregulates transcription of hla, and is an example of an environmental parameter that affects toxin production independently of agr and sarA. How environmental signals are transduced to control alpha-haemolysin and serine protease production, activity and stability at multiple levels are discussed.

Investigation of the SAR of the lead (methoxyalkyl)thiazole 1-[3-(naphth-2-ylmethoxy)phenyl]-1-thiazol-2-ylprop yl methyl ether (1, ICI 211965) led to the methoxytetrahydropyrans, a new series of 5-lipoxygenase (5-LPO) inhibitors exemplified by the parent compound 4-[3-(naphth-2-ylmethoxy)phenyl]-4- methoxy-3,4,5,6-tetrahydro-2H-pyran (4f). In vitro 4f inhibited leukotriene C4 (LTC4) synthesis in zymosan-stimulated plasma-free mouse macrophages and LTB4 synthesis in A-23187-stimulated human whole blood (IC50s 0.5 nM and 0.07 microM, respectively). In the rat 4f inhibited LTB4 synthesis in blood ex vivo and in zymosan-inflamed air pouch exudate with an ED50 3 h after oral dosing of 10 mg/kg in each system. In seeking more potent orally active compounds, strategies were explored in congeners of 4f for reducing lipophilicity without sacrificing potency. For example, replacement of 2-naphthyl of 4f by various aza- and oxoheterocycles afforded compounds in which log P is reduced by 1.7-2.3 units while potency in human whole blood in vitro was maintained or enhanced relative to 4f. In addition, the oxoheterocyclic replacements provided compounds with improved oral potency and the preferred compound from this group is 6-[[3-fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4- yl)phenoxy]methyl]-1-methylquinol-2-one (4y). In the in vitro systems, 4y inhibited LT formation with IC50s in mouse macrophages and human whole blood of 3 nM and 0.02 microM, respectively. 4y did not inhibit the synthesis of cyclooxygenase (CO) products at concentrations up to 500 microM in human blood, a selectivity for 5-LPO over CO of greater than 20,000-fold. In the rat 4y inhibited the formation of LTB4 in blood ex vivo and in inflammatory exudate with ED50s 3 h after oral dosing of 0.9 and 0.3 mg/kg, respectively. 4y was more potent in vitro in human whole blood and in rat blood ex vivo at 3 h than either the 5-LPO inhibitor A-64077 or the FLAP antagonist MK-886. Based on these data 4y (ICI D2138) has been entered into development as an orally active, selective 5-LPO inhibitor for clinical evaluation in inflammatory conditions in which LTs are believed to play a role.

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