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The role of Physiological Factors in the Regulation of Toxin Production by Staphylococcus aureus - FS1524

Toxin production by the food posioning and human pathogenic bacterium Staphylococcus aureus occurs primarily in response to changes in physiological factors at the onset of the postexponential growth phase. Three components which coordinately regulate toxin production have been identified but the physiological stimuli to which they respond and how these signals are transduced into changes in toxin gene exprssion are unknown. The main objective of this proposal is to identify the physiological factors which lead to toxin production and isolate the components of the signal transduction and toxin gene expression control mechanism. This will allow the rational design of procedures to limit toxin production, either by the control of environmental conditions or by the addition of specific inhibitors of the signal transduction pathway.
1) Construction of reporter gene fusions. Stable, Staph, aureus, chromosomal, B-galactosidase, transcriptional fusions in key representative genes involved in toxin production/pathogenicity will be constructed. The fusions will be made in hla, spa ans sar. All of these loci have been cloned and sequenced. I am reisolating the sarA gene from a ZAP Express libary of Staph. aureus 8325-4 chromosomal DNA by hybridisation of an oligonucleotide probe based on the published sarA sequence. 2) The effect of physiological conditions on reporter gene fusion expression will be determined and compared to existing agr:lacz fusion. Postexponential phase regualtion of toxin production of toxin production may in some part be due to a density dependent phenomenon analogous to the Gram negative quorum sensing mechanism mediated by an extracellular factor and htis can be directly tested using the fusions. 3) Identification of components involved in the regulation of toxin production. The agr, sar and xpr loci have already been identified as being involved in the control of toxin production , whether these are all the components which res pond to environmental stimuli and physiological conditions and how they interact to transduce these changes in toxin g ene expression is still unknown. Isolation of transposon mu tants of the fusion strains, under different conditions, wi th altered B-galactosidase expression wil identify genes whose products have either a positive or negative role in toixn production. By the use of 3 different fusions and the existing agr fusion the whole range of componenst involved in the regulation of toxin production should be identified. 4) Genetic analysis of toxin rpoduction regulatory mutants. The number of independent loci identified by transposon mut agenesis will be determined by physical mapping of the tran sposon inserts on the Staph. aureus chromosome. Representat ive mutants will then be chosen and the chromosomal DNA fla nking the transposon insert recovered by subcloning. the in tact loci wil be isolated from a libary of chromosomal DNA by probing with the subcloned fragments. DNA hybridisation will determine the total number of loci involved and identi fyy those which have already been isolated. 5) Biochemical analysis of the toxin production regulatory cascade. All the mutants in independent loci will be screen ed for their effect on the other B-galactosidase fusions an d putative virulance determinant expression such as lipase and B-hemolysin. Biochemical complementation wil determine if an extracellular factor is involved in cell signalling w hich leads to toxin production. This work will therfore beg in to elucidate the hierachy of signal response and control components and how they interact. Major elements can then b e identified as targets for treatments to prevent toxin pro duction. 6) Molecualr biological analysis of toxin regulatory elemen ts. The new cloned loci will then be DNA sequenced in oeder to further understand their genetic organisation and the possible function of their products. 7) Further work. Dependent on results of above. Once the elements of the signalling and regulatory cascade have been identified the molecular interactions which transduce physi ological stimuli into changes in toxin gene expression can be studied.
Time-Scale and Cost
From: 1995

To: 1998

Cost: £144,108
Contractor / Funded Organisations
Sheffield University, Molecular Biology