Srd1p And Rim9p On Adaptation To PH Stress In C.albicans

Candida albicans is the major opportunistic fungal pathogen of humans, and has been gained more attention due to its high incidence of life-threatening infection recently. To survive and proliferate, microorganisms must adapt to various stresses. Extracellular pH represents an important factor influencing cell growth and virulence. The ability of C. albicans to sense and respond to alkaline environments is controlled in a large part by the Rim101pathway. Rim9protein plays a sensor role in that pathway. Multi-protein sequence comparisons suggest that Rim9and Orf19.1510/Srdl proteins play apparently analogous, both of which are members of the Sur7family and contain a Sur7domain. Thus investigation of the Srdlp phenotypically characterization in C. albicans is necessary.ObjectiveThe SRD1and RIM9deletion mutant strains were constructed to investigate whether Srdlp has the similar function to Rim9p, and whether it belongs to Rim101p pathway or not. Array based technologies were used to understand the mechanism of the difference in sensitivity to stresses of two variant strains. In addition, we investigated the effect of heterologous auxotrophy markers from non-C. albicans yeasts (C. maltosa LEU2, C. dubliniensis HIS1and C. dubliniensis ARG4) on adaptation to various stresses of knockout mutationts in C. albicans.MethodsMulti-protein sequence alignments were performed using the MAFFT web application. The entire coding sequence of SRD1and RIM9genes were deleted respectively from the wild type strain SN152by two-step homologous recombination using a fusion-PCR-based Hisl-Leu2-Arg4strategy. Reconstituted strain of SRD1and RIM9were constructed by the same way. The C. albicans SAT1-flipper cassette was used to generate the srd1/rim9double mutant. The SAT marker was subsequently looped out of the final double mutant strain by YPM medium selection. Proper integration of all the target constructs was confirmed by genomic PCR. Spot assays were used to assess the sensitivities of different strains to different stresses, including pH stress, oxidative stress, antifungal agents, metal ions, cell wall stress, osmotic stress and DNA damage agents.10%inactivated FBS, Spider, Lee’s and M199medium were used to assess the hyphal induction. Azole sensitivities of CaLY188by microdilution antifungal susceptibility testing and time-kill curve testing were compared. Vacuole morphology in yeast cells were visualized using the fluorescent dye FM4-64.Genome plasticity is a hallmark of C. albicans. It is proposed that C. albicans generate large-scale genetic variation as a means of adaptation. In this study, we used array based technologies, in combination of array comparative genomic hybridization and gene expression profiling to understand the mechanism of the difference in sensitivity to stresses of two variation strains. These results were confirmed by Q-PCR.To investigate the effect of heterologous auxotrophy markers on adaptation to various stresses of knockout mutations in C. albicans, recombination fragment was constructed by the fusion PCR strategy, which was then used to transform SN152by lithium acetate method. Genomic PCR was used to confirm the integration of those three selectable markers. Spot assays were used to assess the sensitivities of different reintegrated strains to different stresses.ResultsThe results of agarose gel electrophoresis showed that srdl and rim9mutant strains were successfully constructed respectively. Spot assay appears that no particular growth deficiency could significantly differentiate the srdl mutant from the parental strain when the strains were treated with those stresses. However, while loss of Rim9p in C. albicans has significant effects on the cellular response to alkali stress. The rim9mutants failed to form filaments around the colonies in Lee’s medium, spider medium or M199medium. The deletion of SRD1did not affect the morphogenesis change of the rim9single knockout cells. The long C-terminal tail of Srdlp had no effect on ambient pH signaling, either. Neither Rim9nor Srdl protein influences vacuole biogenesis. We also found the rim9mutant was sensitive to amphotericin B and NaCl, but resistant to fluconazole, ketoconazole, H2O2, HU, CFW and Conge Red.Two variant strains were obtained when SRD1gene was knocked out, named as CaLY55and CaLY188. CaLY55showed highly increased sensitivity to many stresses. CGH analysis failed to identify any large segmental variations. In contrast, genomic expression profiling revealed that there were400genes differentially expressed, which were involved in pathogenesis; response to stimulus; oxidation-reduction process; carboxylic acid, oxoacid, and amino acid metabolic process.The resistance to azole antifungal drug of CaLY188by microdilution antifungal susceptibility testing and time-kill curve testing was confirmed. Both CGH and Q-PCR results showed copy number amplification of chromosome R in CaLY188. It got one more copy than the wild-type strain. Its SRD1reconstituted strain CaLY350had the consistent results. Genomic expression profiling in CaLY188revealed250differential genes,76genes (nearly30%of all) located in chromosome R; this could be due to genomic DNA variations. These results could be interpreted that, the resistance to azole phenotype was related to the copy number amplification of chromosome R.The results of agarose gel electrophoresis showed that seven reintegrated strains containing C.m. LEU2, C.d.HISl and Cd.ARG4were successfully constructed respectively. The results of spot assay showed that there was no growth difference between the seven reintegration strains and SN152on response to the tested stresses, including pH stress, oxidative stress, antifungal agents, metal ions, cell wall stress, osmotic stress and DNA damage agents, while only strains which integrated C.d.HIS1marker could survive from the0.02%SDS stress.ConclusionDeletions of the SRD1did not create any significant stress response phenotype in C. albicans, nor did the deletions enhance any of the RIM9deletion effects when combined in a double mutant. Furthermore, challenges in C. albicans show Rim9p but not Srdlp is important for proper response and hyphal formation. RIM9deletion is defective in forming hyphae not only in alkaline media (M199), but also in neutral induction medium (Lee’s, Spider).CaLY55has significant effects on response to various stresses. There is no variation in CGH array.50%of400genes differentially expressed were known associations to stress, which considering that CaLY55was stress responses deficiency in non-stressful growing conditions. Five genes are transcriptional repressors of the hyphal, which contribute to hyphal formation deficiency in CaLY55.The research about CaLY188could be interpreted that the resistance to azole phenotype was related to the copy number amplification of chromosome R. ERG25may be the key, which is role in ergosterol biosynthesis pathway and located in chromosome R.The selectable markers C.m.LEU2, C.d.HISl and Cd.ARG4have no effect on adaptation to most stresses of C. albicans, in which cases SN152could be used as parental control. While assessing the sensitivity to SDS, strains must keep consistency on the existence of CdHISl.