Decline In Mitochondrial Respiratory Function And Fluconazole Resistance In Candida Albicans

More recently, it has been widely accepted that Candida albicans can exploit several cellular responses to facilitate tolerance of antifungal agents and can further acquire resistance by multiple mechanisms. The response to oxidative stress is considered to contribute significantly to modulation of fluconazole (FLC) tolerance in C. albicans in response to antifungal stress. We previously presented evidence that a protective metabolic shift due to mitochondrial respiration deficiency may enable the FLC-resistant C. albicans strain to survive drug challenge partly due to a reduction in generation of intracellular reactive oxygen species (ROS). As a result, it is expected that mitochondrial functions that regulate metabolic behaviour are likely to contribute to fitness and flexibility of C. albicans strains in response to external challenges.Goal To explore the antifungal susceptibility of C. albicans under conditions of mitochondrial respiration blockages and mitochondrial respiration enhancers. To investigate the function of Ald5 in C. albicans, a potential mitochondrial-function-related protein probably encoding an aldehyde dehydrogenase. To identify the potential point mutations in ERG3, ERG11, and TAC1 genes in the matched pair of FLC-resistant and FLC-sensitive C. albicans strains. To characterize and identify the unigenes of ORF19.1510, ORF19.3216, and ORF19.5518 in C. albicans.Methods (Ⅰ) In order to investigate whether cyanide or SHAM would affect drug susceptibility in C. albicans, we used spot and filter disk assays to test C. albicans both clinical and laboratory strains in the presence of cyanide or SHAM. To evaluate possible differences in azole tolerance conferred by Aox1a and Aox1b activity, we compared azole sensitivities of four strains - the WT strain, the isogenetic aox1a/aox1a mutant strain, the aox1b/aox1b mutant strain, and the aox1a/aox1a aox1b/aox1b double mutant strain - by the filter disk method with the treatment of cyanide or SHAM. Alteration of ROS production as measured through DCF fluorescence over time was measured for these four strains. We explored the in vitro combination use of FLC and SHAM against clinical C. albicans isolates by using filter disk and microtiter plates methods. (Ⅱ) To investigate the function of ALD5, we constructed the ald5 null mutant using wild-type strain CAI4. Replacements of the ALD5 alleles with linear disruption fragments were monitored by PCR and southern-blotting with genomic DNA. Then, the susceptibility testing, endogenous ROS production, hyphae induction, biofilm induction, and the interaction with macrophage cell assays were investigated with the wild-type and ald5 null mutant. (Ⅲ) Point mutations in ERG3, ERG11, and TAC1 genes were further examined in the matched pair of C. albicans strains, y0109S and y0109R. (Ⅳ) We performed gene disruption of ORF19.1510, ORF19.3216 and ORF19.5518 in C. albicans using fusion PCR and heterologous markers. First, two pairs of primers were used to amplify genomic DNA on the 5'side and 3'side of the target gene separately. Another pair of primers was used to amplify the selectable markers C. dubliniensis HIS1 and C. maltose LEU2 separately. The first round of gene disruption was carried out with C. dubliniensis HIS1, and the second round was carried out with C. maltose LEU2. After selection of transformants on the appropriate single or double amino acid dropout medium, gene disruption candidates were screened by PCR for expected 5'and 3'junctions as well as the size of the disrupted gene. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. Then, morphosis, the susceptibility testing, and the cell cycle analysis were investigated with the wild-type and the target genes null mutants.Results (Ⅰ) The addition of 1 mM cyanide to the culture medium resulted in decreased susceptibility to FLC, itroconazole (ITC), ketoconazole (KTC), and miconazole (MCZ) for both FLC-resistant and -sensitive C. albicans strains. The drug sensitivity was further confirmed for these four strains by the filter disk method, in which the diameters of the inhibition zones of FLC, ITC, KTC, and MCZ disks were significantly diminished in the presence of 1 mM cyanide. Meanwhile, all four strains were strikingly susceptible to the four azoles tested in the presence of 5 mM SHAM, with larger inhibition zones than those in the absence of SHAM. Furthermore, with the addition of 1 mM cyanide, the wild-type and the isogenetic aox1a/aox1a mutant strain strains with the induction of the Aox1b showed hyper-tolerance to all azoles tested, whereas the degree of reduced sensitivity to azole antifungals for aox1b/aox1b mutant strain with the Aox1a induction was less evident. With the treatment of the ROS-inducing agents, MCZ and benomyl, ROS generation was augmented in the aox1a/aox1a mutant, aox1b/aox1b mutant, and aox1a-aox1b/aox1a-aox1b mutant, to a greater degree than in the wild-type strain. The addition of SHAM remarkably reduced the MIC80s of FLC, as was evident from the checkboard analysis and filter disk assay. FLC MIC80s against resistant isolates decreased more than 128 fold, and the MIC80s against susceptible isolates decreased from 16 to 25 fold. Among the 20 clinical isolates, synergy between FLC and SHAM was observed in 17 (85%), and indifference was observed in 3 (15%) susceptible isolates. (Ⅱ) We successfully constructed the ald5 null mutant in C. albicans. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in cells. The results showed that the ald5 null mutant has no significant difference in forming hyphae in comparison to the wild-type strain. The susceptibility of the ald5 null mutant to macrophage cells, hydrogen peroxide, salt, and osmotic stresses was similar to it of the wild-type strain. In the non-fermentation medium, the ald5 null mutant grew slowly, while had no cell cycle arrest. The ald5 null mutant showed decreased susceptibility to azoles in a certain degree in the non-fermentation medium. (Ⅲ) For the strains y0109S and y0109R, the only one missense mutation in ERG3 gene was the single amino acid substitution D19E. Meanwhile, both strains harbored one nucleotide change at base position 304 (ACC) in ERG3 which had no effect in the amino acid sequence. There were no mutations in TAC1 in the strain y0109S. In contrast, TAC1 gene from the strain y0109R was showed a total of two missense mutations (L47K and N977K) in comparison with the published sequence dataset. In addition, there was no amino acid difference in Erg11 in these two strains. (Ⅳ) We performed the disruption of ORF19.1510, ORF19.3216, and ORF19.5518 using a two-step fusion PCR and heterologous markers. The disappearance of the product of the internal check primer pairs confirmed the complete disruption of target genes. The orf19.1510 null mutant showed a changed budding pattern: in the middle-exponential phase, a small proportion of the orf19.1510 null mutant was unusually enlarged, and with random budding scars; in the stationary phase, most of the orf19.1510 null mutant cells were the same size as the wild-type strain, but there were still a proportion of cells that were enlarged, round, and with random budding scars. The orf19.1510 null mutant had a small effect on germ tube formation in YPD containing 10% FBS liquid culture at 37℃, and no hyphal growth was seen in spider or Lee's medium, either on solid or in liquid culture at 37℃. The orf19.1510 null mutant was sensitive to azoles, sodium chloride, and double-strand DNA damage agent, phleomycin (PHL), but was resistant to single-strand DNA damage agents, methyl methane sulfonate (MMS), and hydroxyurea (HU). The orf19.1510 null mutant has the same sensitivity to peroxide oxygen and sorbitol. The DNA contents of many more single cells of the orf19.1510 null mutant were in 4N (G2) phase in the early-, middle-log growth phase. The DNA contents of a majority of single cells of the orf19.1510 null mutant in the stationary phase were in between the 2N (G1) and 4N (G2) phases. The ORF19.3216 and ORF19.5518 null mutants showed no difference in the phenotypes in our study.Conclusion (Ⅰ) Our present study indicated that alternative, cyanide-insensitive, respiration in C. albicans leads to azole tolerance through the reduction of intracellular ROS production. This is a novel mechanism contributing to decreased FLC susceptibility and increased survival of C. albicans. We showed in this study that cyanide treatment, with the resulting induction of the alternative pathway in C. albicans, caused reduced susceptibility to antifungal azoles, and that the inhibition of alternative respiration by SHAM resulted in enhanced susceptibility to azoles. By testing the aox mutant C. albicans strains, we identified that Aox1b played a major role in conferring azole tolerance. Our analysis of intracellular ROS formation further proved that the Aox in C. albicans lowered mitochondrial ROS levels upon antifungal drug treatment. These findings provide a novel mechanistic insight into FLC tolerance in C. albicans and explain how the alternative oxidative pathway might regulate electron flux and mitochondrial respiration and thereby generate a survival advantage during antifungal stress.(Ⅱ) We successfully constructed the ald5 null mutant in C. albicans and showed that the deletion of ald5 decreased susceptibility of C. albicans to azoles in a certain degree in the non-fermentation medium. We proposed that ALD5 played an important role in the production of mitochondrial NADPH, which contributed to increasing the ROS level in C. albicans cells.(Ⅲ) We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hyperactivity of Tac1 could up-regulate CDR1, CDR2, and PDR17 in C. albicans and result in resistance to azole. A single amino acid difference (D19E) in ERG3 that led to inactivation of Erg3 could accumulate sterol precursors along with changes in expression of ergosterol biosynthesis genes, which could contribute to azoles resistance in C. albicans.(Ⅳ) We successfully constructed the orf19.1510 null mutant, orf19.3216 null mutant, and orf19.5518 null mutant of C. albicans. The results showed that the deletion of ORF19.1510 significantly increased susceptibility of C. albicans to azoles, sodium chloride, and double-strand DNA damage agent (PHL), but decreased sensitivity to single-strand DNA damage agents (MMS and HU). The deletion of ORF19.1510 was deficient in the yeast to hyphal switch. We proposed that the primary effect of ORF19.1510 depletion is accumulation of unrepaired DNA damage and the subsequent activation of the DNA-damage checkpoint. One of the activated molecules triggers the phenotypes. The deletion of ORF19.3216 and ORF19.5518 had no effect in the phenotypes in current study. Further experimental investigations are needed.