In the pathogenic yeast Candida albicans (Ca), clinical isolates are commonly analyzed for their growth response to a gradient of growth responses. These growth patterns are the result of gene expression patterns within the cell. In resistant isolates, four genes are known to be constitutively overexpressed include the target enzyme ERG11, and three efflux pumps, CDR1, CDR2, and MDR1. In susceptible Ca, these genes can be transcriptionally induced when exposed to drugs. However, the response to azole by these genes has not been analyzed in resistant strains. This proposal is based on the hypothesis that the cell's response to drug may be the result of altered expression of known and unknown resistance genes that includes constitutive overexpression in the absence of drug and induced gene expression in the presence of drug. The overall goals of this proposal are to define and characterize the response to antifungal drug for a spectrum of clinical isolates, and to investigate those isolates for altered gene induction as a mechanism of resistance apart from constitutive overexpression.
The Specific Aims of the proposal as follows:
Aim 1 - To define the spectrum of responses of fungal cells to azoles. Clinical isolates will be clustered based on their growth responses to azole drugs. Eight representative isolates from these clusters will then be analyzed in subsequent aims for gene expression.
Aim 2 - To identify unique gene induction patterns using expression profiling. Representative strains from the cluster analysis in Aim 1 will be expression profiled using microarrays to identify and characterize the gene induction patterns in response to a gradient of seven drug concentrations.
Aim 3 - To characterize unique azole-induced gene expression patterns. QRT-PCR will be used to characterize the induction patterns of specific genes in detail.
Aim 4 - To identify and characterize the molecular mechanisms associated with altered expression patterns. Mutations in the clinical isolates will be identified by sequencing and then characterized as reconstructions in laboratory strains. This proposal focuses on how azoles alter the cell's growth and how azoles alter the patterns of gene induction, defining a dynamic response of fungi to azole drugs. This has clinical significance, as fungi in a patient are never simply exposed to a single high drug concentration. The results from this study will have implications for the development of new therapies and new diagnostics of resistance, and for restructuring therapies to maximize current antifungal treatment.
The pathogenic yeast Candida albicans is the most common opportunistic infection in AIDS patients, occurring in over 90% of patients. Previous analyses of Candida's response to azole drugs and Candida drug resistance have used susceptible and resistance clinical isolates. Gene expression analysis in these isolates has focused on constitutive gene expression in the absence of drug, and on gene expression at a single drug concentration. The current proposal will focus on gene induction over a select spectrum of conditions to understand the dynamic response of fungi to azole drugs. This has clinical significance, as fungi in a patient are never simply exposed to one defined set of drug/fungus conditions.
| Brown, Gordon D; Denning, David W; Gow, Neil A R et al. (2012) Hidden killers: human fungal infections. Sci Transl Med 4:165rv13 |
