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A study of the importance of dityrosine in Aspergilli using a bioinformatic and molecular biological approach

The staggering ability of microbes to acquire multiple systems to reduce or avoid the action of antibiotic treatments and the limited number of novel antibiotics identified through random screening, alongside the increasing number of immuno-compromised patients requiring treatment has led to a more directed approach in the search for novel antibiotics.  This has been made possible by the availability of complete genome sequences of many micro organisms allowing a drug-target directed approach.

The fellowship project combines the knowledge that a group of proteins: the Cytochrome P450s (CYP) are involved in important biosynthetic reactions and that inhibitors to some have been developed as commercially important drugs; e.g. the commonly used azole antifungal agents.  By using bioinformatics homology of CYP genes that have known function for cell survival and growth in one organism this may lead to the discovery of a potential drug target in another.

Thus a CYP sequence found in Saccharomyces cerevisae which is involved in the biosynthesis of dityrosine a major component of the spore cell wall surface is CYP56 or DIT2.  A similar gene sequence has been identified in Aspergillus nidulans using bioinformatics and furthermore there is evidence of another gene with homology to DIT1 from S.cerevisae.  The fellowship project will start by confirming the presence of these homologous sequences in A.nidulans, the genes in A.nidulans will be cloned into plasmids for transformation into a strain of S.cerevisae in which the native DIT1 and DIT2 genes have been ‘knocked out’.  If the A.nidulans genes are expressed in the transformed yeast and fulfil the same function then the yeast will still produce dityrosine.

The second half of the fellowship will involve checking for homologous genes in A.fumigatus the pathogenic organism which causes lung infections - aspergillosis; the protein of DIT2(CYP56) could be modelled as a way of investigating  potential drug- protein interaction and  transcriptional analysis would provide insights with respect to timing of therapy in view of pathogen life cycle.

The proposal will provide complete training in the molecular biological techniques necessary to follow through from gene identification to protein synthesis; the use of biochemical procedures to identify metabolites produced and an understanding of the increasing importance of the role of bioinformatics in drug discovery.