Profiling gene expression inCoccidioides posadasii

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.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection

“…Fungal rhodopsins and opsin-related proteins have been recently identified in several fungal genomes as homologues of bacteriorhodopsin, with unknown function (reviewed by Brown, 2004). In the human pathogenic fungus Coccidioides posadasii, the opsin-1 gene has also been found to be specifically expressed during parasitic growth of the fungus, but not during saprobic development (Delgado et al, 2004). Genes encoding key enzymes of the glyoxylate cycle were also observed to be activated in A. benhamiae during infection, i.e.…”

Agrobacterium -Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor

“…Using Blastn against the GenBank EST database, a 59-nt region of clone Ma#267 showed 76% identity to three ESTs (GenBank accession numbers CO035825, CO032944, and CO021837) from both saprobic and pathogenic spherule cDNA libraries of Coccidioides posadasii (17,41). These ESTs form a contig of 1,558 nt that is 98% identical over the same length to the mRNA of GenBank accession no.…”

Phase-specific gene expression underlying morphological adaptations of the dimorphic human pathogenic fungus, Coccidioides posadasii

“…The ability to induce both growth forms under laboratory conditions makes it possible to identify phase-speciWc genes. By using suppression subtractive hybridization (Diatchenko et al, 1999), Delgado et al (2004) identiWed four genes whose expression is essentially restricted to the parasitic cycle. The use of DNA microarray technology to proWle gene expression has enabled further identiWcation of candidate genes that may be vital to the development of particular phases of the life cycle of pathogenic fungi, and enabled the analysis of global patterns of gene expression between developmental phases (e.g., Hwang et al, 2003).…”