RESUMO
Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species.
Assuntos
Adaptação Fisiológica/genética , Candida/genética , Candida/fisiologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Interações Hospedeiro-Patógeno/genética , Animais , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/patogenicidade , Parede Celular/química , Parede Celular/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Regulação Fúngica da Expressão Gênica , Humanos , Larva/microbiologia , Leucócitos Mononucleares/imunologia , Macrófagos/microbiologia , Mariposas/microbiologia , Estresse Fisiológico/genética , VirulênciaRESUMO
Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii.