Cloning of Aspergillus fumigatus histidine kinase gene fragment and its expression during invasive infection.

Abstract

It was demonstrated recently that three histidine kinases genes in Candida albicans contributed to virulence, indicating the importance of signaling pathways regulated by histidine kinases. In the present study, using a set of degenerate primers, RT-PCR was performed with cDNA of A. fumigatus as a template. PCR products were cloned and sequenced. After Blast analysis, it was found that one fragment (named as AFHK1), 305 bp, was highly homologous to the two-component histidine kinase tesA gene of Aspergillus nidulans. But AFHKI was not completely identical to the FOS-1 gene of A. fumigatus. The same A. fumigatus strain was used to inoculate the mice for a murine model of invasive pulmonary aspergillosis (IPA). After 5-days post-inoculation, the lungs of infected animals were removed and incubated for 2 h at 37 degrees C in digestion buffer containing collagenase and trypsin. The pulmonary cells were removed by passing the suspension through a sieve. The non-filterable hyphae were treated with deoxygenated sodium cholate. Total RNA of A. fumigatus isolated from the infected tissues or cultured in vitro was extracted. With AFHKI as a probe. a Northern blot was performed. A 3.0 kb (approximate) transcript of mRNA was detected corresponding to the putative histidine kinase gene. It was demonstrated that that gene was expressed at markedly higher levels in vivo than in vitro. The results suggest that this gene may contribute to the survival and virulence of A. fumigatus.