New strategies for antifungal development in Cryptococcus neoformans exploiting purine metabolism and microevolution during infection

Carl Morrow (2011). New strategies for antifungal development in Cryptococcus neoformans exploiting purine metabolism and microevolution during infection PhD Thesis, School of Chemistry & Molecular Biosciences, The University of Queensland.

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Author Carl Morrow
Thesis Title New strategies for antifungal development in Cryptococcus neoformans exploiting purine metabolism and microevolution during infection
School, Centre or Institute School of Chemistry & Molecular Biosciences
Institution The University of Queensland
Publication date 2011-10
Thesis type PhD Thesis
Supervisor James Fraser
Bernard Carroll
Total pages 227
Total colour pages 36
Total black and white pages 191
Language eng
Subjects 060107 Enzymes
110802 Medical Infection Agents (incl. Prions)
060503 Microbial Genetics
Abstract/Summary Cryptococcus neoformans is one of the leading causes of opportunistic fungal infections in immunocompromised individuals worldwide. Currently available antimycotic agents target a limited repertoire of fungal-specific cell wall or membrane components, and have high toxicity, poor efficacy and are few in number. As Cryptococcus is responsible for over 625,000 deaths annually within a growing cohort of susceptible individuals, particularly the AIDS population, the need for new agents to target this growing threat is vital. To address this issue of limited treatment options in fungi, a dual approach was taken to identify new strategies for antifungal development in Cryptococcus. Firstly, a historically productive source of antiproliferative therapies, the purine metabolic pathway, was examined and a specific candidate protein, IMP dehydrogenase (IMPDH) was characterised in particular detail. Secondly, a more holistic, unbiased method was used to identify novel targets by examining the process of microevolution in the genomes of serial clinical isolates of Cryptococcus, a phenomenon commonly observed in pathogenic fungi. Key enzymes involved in GTP biosynthesis and salvage in Cryptococcus, IMPDH and hypoxanthine xanthine guanine phosphoribosyltransferase, respectively, were initially identified and characterised. Mutants in the salvage enzyme displayed no phenotype except in the presence of inhibitors of IMP dehydrogenase, and were unaffected for virulence in animal models. In contrast, IMPDH mutants are auxotrophs, yet exhibit a variety of phenotypic defects even when supplemented with salvageable substrates. Notably, strains deficient in the enzyme are avirulent in animal models and are cleared rapidly from the lungs. Cryptococcus is particularly sensitive to the widely utilised IMPDH inhibitor mycophenolic acid, although a rare subtype that possesses a naturally resistant isoform was identified. Extensive protein mutagenesis revealed that a combination of two residues near the active sites is required for resistance, in a strikingly similar fashion to a distantly related IMPDH from a highly MPA resistant protozoan parasite. Functional studies of the two enzymes suggested they possess a similar kinetic profile to the other major fungal pathogen Candida albicans, in which IMPDH has been proposed as a drug target. The structure for both proteins in complex with substrate and inhibitor was solved by X-ray crystallography, which will guide future structure-based drug design efforts. Crucially, addition of IMP dehydrogenase inhibitors was found to be protective against Cryptococcus infection in an animal model, validating the approach. Existing genome data from an intensively studied isolate of Cryptococcus was next explored for structural variations that may influence pathogenicity. Synteny analysis of the clinically dominant C. neoformans var. grubii genome in comparison with two less clinically prevalent subtypes revealed it harbours a surprisingly limited number of large genome rearrangements. Six inversions and one duplication were identified which are unique to the var. grubii clade, all which appear to have arisen recently. In addition, a large translocation was identified in the genome strain only, which disrupted two genes of unknown function. Gene knockouts in a related strain showed they affected three key cryptococcal virulence factors, although overall virulence is unaffected in animal models. One of the genes is involved specifically with the metabolism of glucose at human body temperature, while the other is a homeodomain-containing transcription factor of unknown function. To identify selective pressures occurring in vivo that may be new antifungal targets, a panel of serial Cryptococcus isolates taken over the course of an infection were analysed phenotypically and their karyotypes mapped using a Southern hybridisation strategy. Aneuploidies, duplications and translocations were all identified in these strains during mitotic cell growth within the human host. All strains except one displayed regular changes in the size of the rDNA repeat, which may represent a concerted strategy during infection. Notably, a region on the left arm of chromosome 8 was found in variable copy number in three separate isolates, while another strain developed an isochromosome of the left arm of chromosome 12 from an initial chromosome 12 aneuploidy. This latter strain was extensively characterised, revealing profound differences in phenotype, virulence components and pathogenesis during infection in a mammalian model. Generation of karyotypic variation may be a common strategy during cryptococcal infection, and further analysis of isolates may reveal trends in gene duplications that present attractive drug targets. The contrasting approaches adopted in this work provide promising results and present multiple avenues for further study and chemotherapeutic agent development. The comprehensive genetic, structural and functional characterisation of IMPDH in conjunction with in vivo virulence studies establishes a robust foundation for development and optimization of fungal-specific inhibitors in de novo GTP biosynthesis, while whole genome sequencing efforts already initiated will precisely characterise genomic changes occurring in strains evolving under long term selection. Unconventional approaches such as these will be required to more effectively treat the increasing burden of life-threatening systemic fungal infections.
Keyword Cryptococcus neoformans
Cryptococcus gattii
pathogenic fungi
chromosomal rearrangement
GTP biosynthesis pathway
IMP dehyrogenase
Additional Notes 65, 67, 69, 71, 72, 74, 76, 101, 102, 109-113, 122, 137, 139, 144, 147, 151-155, 170, 187-189, 191-194, 196, 198, 200, 202

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Created: Mon, 23 Apr 2012, 16:13:05 EST by Carl Morrow on behalf of Library - Information Access Service