NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

Abstract

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

The role of AtfA and HOG MAPK pathway in stress tolerance in conidia of Aspergillus fumigatus

“…This fragment was fused into pPTRI using the GeneArt system, resulting in pPTRIatfA+, which was used for the transformation of DatfA to construct the ectopically complemented strain Co-atfA. A. fumigatus transformation was performed according to conventional methods for protoplast-polyethylene glycol transformation for Aspergillus (Hagiwara et al, 2013). Homologous recombination and correct gene replacement were confirmed by polymerase chain reaction (PCR) of the genomic DNA, and the absence of the mRNA of the target gene was verified using realtime reverse-transcription (RT)-PCR analysis.…”

Whole-Genome Comparison of Aspergillus fumigatus Strains Serially Isolated from Patients with Aspergillosis

“…A calculation of the number of conidia obtained was performed as described previously (23). Briefly, conidia on PDA (10 4 conidia/ml, 3 ml per well) were incubated at 37°C in 6-well plates.…”

Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

“…A. fumigatus strain Af293 and the srbA deletion mutant were used for expression analysis. The strains were cultivated in 0.1% yeast extract containing glucose minimal media (YGMM) at 37°C (56). Itraconazole (Wako Pure Chemical Industries) and amphotericin B (Sigma-Aldrich Co) were commercially obtained.…”

“…These flanking regions and the ptrA marker were fused into pUC119 using the GeneArt system (Invitrogen), resulting in a plasmid pUC119-srbA::ptrA, which was used for transformation to construct the ⌬srbA mutant. A. fumigatus transformation was performed according to conventional methods for protoplastpolyethylene glycol transformation for Aspergillus as performed previously (56). Homologous recombination and gene replacement were confirmed by PCR of the genomic DNA, and the absence of mRNA of the target gene was verified using RT-PCR.…”

“…RT-PCR was performed using the 7300 system (Life Technologies Corp.) with SYBR green detection as described previously (56). Briefly, the Thunderbird SYBR qPCR mix was used for reaction mixture preparation (Toyobo).…”