Differential involvement of histidine kinase receptors in pseudohyphal development, stress adaptation, and drug sensitivity of the opportunistic yeast Candida lusitaniae

Abstract

Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast Candida lusitaniae. We isolated three genes, SLN1, NIK1, and CHK1, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the "URA3 blaster" strategy. Functional analysis of disruptants was undertaken, except for the sln1 nik1 double mutant and the sln1 nik1 chk1 triple mutant, which are not viable in C. lusitaniae. The sln1 mutant revealed a high sensitivity to oxidative stress, whereas both the nik1 and chk1 mutants exhibited a more moderate sensitivity to peroxide. We also showed that the NIK1 gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the sln1 mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the SLN1 gene could be crucial for the C. lusitaniae yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of SLN1 mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in C. lusitaniae.

FIG. 1.

(A) Structure of the C. lusitaniae HKR proteins. The putative phosphorylable residues are indicated with triangles. EL, extracellular loop; HKcd, histidine kinase catalytic domain; R, repeated sequence; RD, receiver domain; S/TKrd, serine/threonine kinase-related domain; TMH, transmembrane helix. (B) Dendrogram generated after alignment of the predicted sequences of C. lusitaniae HKRs (ClSln1p, ClNik1p, and ClChk1p) with sequences retrieved from C. guilliermondii (CguiSln1p, CguiNik1p, and CguiChk1p), C. tropicalis (CtSln1p, CtNik1p, and CtChk1p), C. glabrata (CgSln1p), and S. cerevisiae (ScSln1p). Alignment utilizes the neighborhood-joining method from TreeView PPC software. Distances along the branches represent the divergence between two cognate sequences.

FIG. 2.

Morphology of C. lusitaniae cells and expression analysis of genes encoding HKRs. (A) Morphology of pseudohyphae emerging from the edge of a colony when cells are plated on YCB solid medium. The morphology of budding yeast cultured in YCB liquid medium is shown in the top left of the picture. (B) Semiquantitative RT-PCR of the three genes encoding HKRs (SLN1, NIK1, and CHK1). A representative RT-PCR analysis is shown. For each target gene, the amount of transcription was compared to that of the ACT1 gene (relative level of transcription). The histogram presents the mean values of results from three independent experiments. Error bars show standard deviation. Wild-type cells were cultured during 48 h in YPD liquid medium (lane 1), in YPD solid medium (lane 2), in YCB liquid medium (lane 3), or in YCB solid medium (lane 4). ph, pseudohyphal formation.

FIG. 3.

Southern blot hybridization and schematic representation of resident loci SLN1, NIK1, and CHK1 and of molecular events that occurred in transformants. Signals revealed by the labeled probes (each marked with an asterisk) correspond to those expected from the genomic restriction map. (A) Hybridization pattern with SLN1 and REP probes of XhoI-digested genomic DNA from 6936 ura3_[Δ360] (a) or a representative sln1::GUN transformant (b). (B) Hybridization pattern with NIK1 and REP probes of PstI-digested genomic DNA from 6936 ura3_[Δ360] (a) or a representative nik1::GUN transformant (b). (C) Hybridization pattern with CHK1 and REP probes of SacII-digested genomic DNA from 6936 ura3_[Δ360] (a) or a representative chk1::GUN transformant (b). DNA fragment sizes are indicated in kilobases.

FIG. 4.

Measurement of cell survival after oxidative stress imposed by the addition of 2 mM or 5 mM H₂O₂ in simple mutants (sln1::GUN, nik1::GUN, and chk1::GUN) and double mutants (chk1::GUN sln1::REP and chk1::REP nik1::GUN). The percent survival is expressed with respect to that of an H₂O₂-treated control sample of the wild-type strain (100%). Standard deviation bars are based on three individual replicates.

FIG. 5.

Susceptibilities of C. lusitaniae mutants to iprodione and fenpiclonil. The wild-type strain and representative simple mutants (sln1::GUN, nik1::GUN, and chk1::GUN), double mutants (chk1::GUN sln1::REP and chk1::REP nik1::GUN), and reintegrant strains (sln1+SLN1, nik1+NIK1, chk1 sln1+SLN1, and chk1 nik1+NIK1) were grown for 48 h on YPD plates containing fenpiclonil or iprodione at concentrations indicated.

FIG. 6.

Pseudohyphal growth abilities of mutants. Cells (10⁶; contained in drops of 5 μl) of the wild-type strain and representative simple mutants (sln1::GUN, nik1::GUN, and chk1::GUN), double mutants (chk1::GUN sln1::REP and chk1::REP nik1::GUN), and reintegrant strains (sln1+SLN1 and chk1 sln1+SLN1) were spotted on YCB solid medium supplemented or not with discriminatory concentrations of sorbitol (1 M), NaCl (0.5 M), KCl (0.5 M), MG (15 mM), and H₂O₂ (1 mM). Observations were done 48 h after spotting. This experiment was done in triplicate, and pictures present representative structures observed for each sample.

FIG. 7.

Delay of pseudohyphal development of the sln1::GUN mutant. The wild-type strain, the sln1::GUN simple mutant, and the corresponding sln1+SLN1 reintegrant strain were simultaneously spotted on YCB solid medium. Observations were done at 3, 6, 12, and 24 h after spotting. This experiment was done in triplicate, and pictures present representative structures observed in each sample.