Abstract
Coccidioides posadasii is a dimorphic fungal pathogen which grows as a filamentous saprobe in the soil and multicellular parasitic form in host lung tissue. Studies of gene expression profiles during saprobic and parasitic phase development can provide clues about morphogenetic regulation and may lead to the discovery of molecular targets for novel antifungal drugs. Suppression-subtractive hybridization (SSH) and quantitative real-time polymerase chain reaction (QRT–PCR) were used to identify and quantify differential gene expression during in vitro growth of Coccidioides. DNA fragments obtained from the subtraction of cDNA pools derived from the saprobic and parasitic phase RNA preparations were each cloned into an appropriate vector and subjected to sequence analysis. Semi-quantitative, reverse transcription polymerase chain reaction (RT–PCR) experiments were first conducted to assess whether these inserts represented differentially expressed genes. Nucleotide sequences of the partial and full-length genes selected by RT–PCR were obtained by genome walking and rapid amplification of cDNA ends (RACE) methods. QRT–PCR analysis of the expression of these genes during saprobic and parasitic cell growth was then conducted using DNA standard curves normalized to a constitutively expressed control gene. Four C. posadasii genes whose expression is essentially restricted to the parasitic cycle were discovered using this approach. These genes include homologues of OPS1 (encodes opsin-related protein), MDR1 (multidrug resistance protein), ALDR1 (aldehyde reductase), and PSP1 (hypothetical lipid transporter/flippase protein). The combined applications of SSH and QRT–PCR permit global analysis of gene expression patterns in C. posadasii.