Thermally regulated gene expression in Penicillium marneffei.

摘要

Penicillium marneffei is an emerging fungal pathogen in Southeast Asia. A dimorphic fungus, it undergoes a reversible, temperature-dependent shift in morphology concomitant with infection. To date, no studies have examined the changes in gene expression that accompany this transition. Here, the identification of several thermally-sensitive genes is described using differential display RTPCR (DD-RTPCR) is described. Several candidate gene fragments were found with enhanced expression at either 25°C or 37°C. These all encoded metabolic genes that encode enzymes involved in nitrogen metabolism, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle (GC). Based on this, it is postulated that survival of P. marneffei at elevated temperature, and in a host, is dependent largely on a stress response.; A gene encoding the enzyme malate synthase (MLS) was upregulated at 37°C. This enzyme, along with isocitrate lyase (ICL), form the key components of the GC. The GC is an anaplerotic pathway used by microbes to survive on certain carbon sources, such as acetate and lipids, in the absence of glucose. Recent studies have described the potential importance of this pathway in the pathogenesis of Mycobacterium tuberculosis and Cryptococcus neoformans . Temperature-enhanced expression of both these genes (PmMLS1 and PmICL1) was confirmed in P. marneffei by Northern analysis. The complete gene sequences for these two genes were obtained from a library of genomic P. marneffei DNA. Comparison of the derived protein sequences showed high homology to the corresponding proteins of other fungi, bacteria, and plants. Several interesting structural features were identified in PmICL1, including two possible regulatory motifs. The promoters of these genes both contain putative binding sites for a homolog Gcn4p, a highly conserved yeast transcription factor involved in response to amino acid starvation. The promoter of PmICL1 also possesses putative binding sites for AbaA, a transcription factor implicated in certain aspects of dimorphism in P. marneffei.; Based on expression data and its potential importance in the pathogenesis of several microbes, the GC may serve as an excellent target for the development of future antifungal agents. The high conservation of these genes across microbes makes it possible that such agents will be broad spectrum.