Cryptococcus neoformans is a major pathogen in immunocompromised patients, causing life-threatening meningoencephalitis in approximately 6% of HIV positive individuals. In vitro melanin production has classically been associated with virulence in C. neoformans. During the previous funding period congenic knockout strains of C. neoformans establish the importance of the CNLAC1 gene in virulence and laccase-dependent catecholamine oxidation products have been identified in the brain of infected mice. Furthermore, a complex pattern of regulatory DNA-binding sites upstream of the CNLAC1 gene have been identified over been identified together with an unusual Sp1 enhancer site. Sp1 consensus sequences have been found in the other virulence genes such as Cap64 and Cap59 suggesting co-regulation of virulence. The hypothesis to be examined is that molecular regulators of CNLAC1 control virulence of Cryptococcus neoformans. The objectives of the proposed research are to identify and characterize genes involved in laccase expression to determine their role in virulence.
Aim 1 proposes to identify and characterize important regulatory DNA-binding sites in the upstream region of CNLAC1. The plan is to use pVEW promoter plasmid and electromobility shift assays to determine significant enhancer and repressor regions under conditions of glucose repression and derepression as well as the host temperature of 37 C.
Aim 2 proposes to identify and characterize genes involved in transcriptional enhancement of repression of CNLAC1 and determine their role in virulence. Genes will be cloned by homology to genes predicted by CNLAC1 DNA-binding sites and from laccase-deficient insertional mutants. For each gene the plan is to produce knockout and wild-type complemented strains of C. neoformans and test the effects of the gene on laccase production, capsule formation, secreted manno-proteins, urease activity and virulence.
Aim 3 will identify and characterize genes involved in post-transcriptional modification of laccase from DL-1 mutants. Initially, the plans are to complement a vacuolar H+-ATPase on laccase secretion and assess for possible co-regulation of CNLAC1 and CNVPH1. It is anticipated that these studies will provide insights into regulation and virulence factors in C. neoformans that may be used to provide novel approaches to the treatment and control of cryptococcosis.
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