Preliminary Study On The Role And Mechanism Of QCR7 Gene In The Pathogenicity Of Candida Albicans

Objective:Candida albicans is an important conditional pathogenic fungus.In recent years,with the increase in the number of patients with various types of tumors,autoimmune diseases,organ transplants and other diseases,as well as the use of various intubation procedures and hormone drugs in clinical treatment,the number of people infected with Candida albicans has increased.It has been reported that the electron transport chain(ETC)of mitochondria plays an important role in the growth,reproduction and morphological transformation of Candida albicans,such as hypha differentiation,biofilm formation,adaptation to external environmental pressure,biosynthesis and structural stability of cell wall,and interaction with innate immune cells.However,the pathogenesis of ETC complex III in Candida albicans is rarely studied.The purpose of this study was to explore the role and mechanism of complex III helper protein subunit QCR7P in the pathogenicity of Candida albicans,so as to provide a basis for further research on the pathogenicity of Candida albicans and the development of novel antifungal drugs.Methods:1.Candida albicans QCR7 gene knockout strain SN152(qcr7Δ/Δ)and complementary strain SN152(qcr7AB)were constructed by homologous recombination gene knockout technique using Candida albicans SN152 as parent strain;2.Balb/c mice were injected into tail vein with solubility of 5×106 CFU/ml SN250,qcr7Δ/Δ and qcr7AB 100 ul,5 mice in each group,respectively.A systemic infection model of mice was established and the survival was observed and recorded to investigate the effect of QCR7 gene on the virulence of Candida albicans;3.The effect of QCR7 gene on the growth of Candida albicans was observed by measuring the growth curves of SN250,qcr7Δ/Δ and qcr7AB cultured in YPD liquid medium for 20hours.The initial solubility OD600 was 0.1;4.Spot assay test was used to explore the growth of qcr7Δ/Δ on various stress media(YPD solid medium supplemented with H2O2,CuSO4,LiCl,NaCl,Congo red and sorbitol,respectively),and meaningful phenotypes were screened.At the same time,Spot assay experiment was used to observe the growth of qcr7Δ/Δ on the medium containing different carbon sources(YEP solid medium with glucose,sucrose,mannitol,glycerol,ethanol and acetate alcohol,respectively);5.At the same time,the liquid medium and solid medium of YPD+10%FBSand Spider were used to induce the mycelium growth of WT and qcr7Δ/Δdelta,to observe the effect of QCR7 gene on the mycelium growth of Candida albicans,while the liquid medium containing different carbon sources(YEP liquid medium with glucose,sucrose,glycerol and ethanol)was used to induce the growth of qcr7Δ/Δ delta mycelium,and the effects on the mycelium growth of qcr7Δ/Δ delta under different induction conditions were observed;6.The in vitro biofilm experiment was carried out in a 96-well polystyrene plate to study the effect of QCR7 gene on the biofilm formation of Candida albicans;7.WT and qcr7Δ/Δ were induced in RPMI1640 liquid medium at 37℃ for 2h and RNA was extracted.Transcriptome sequencing was completed with Illumina Hi Seq platform.Results:1.Identification of QCR7 gene knockout strains:(1)the positive transformants could grow in YPD,SD/-Leu,SD/-His and SD/-Leu-His solid medium for 48 hours;(2)using the positive transformant genome as a template,the nutritional screening marker genes Cm.Leu2 and Cd.Hisl successfully replaced the two alleles of QCR7 by PCR identification.Identification of QCR7 gene complementary strains:using the positive transformant genome as atemplate,the nutritional screening marker genes Cd.Arg4 and QCR7 genes successfully replaced the marker genes Cm.Leu2 or Cd.Hisl by PCR identification;2.In the group of 5 Balb/c mice injected with wild type strain through tail vein,the first one died on the 4th day,and all of them died on the 10th day.No death occurred in the mice injected with qcr7Δ/Δ and qcr7AB within 30 days;3.The results of growth curve test showed that SN250,qcr7Δ/Δ and qcr7AB all entered the logarithmic phase at about 4 hours,qcr7Δ/Δ entered the platform phase at the 11th hour,and the others entered the platform phase at about 12 hours.During the platform period,the OD600 value of SN250 is significantly different from that of qcr7Δ/Δ,while the OD600 value of qcr7AB is between qcr7Δ/Δ and SN250;4.Spot assay results showed that the growth of qcr7Δ/Δ was inhibited on YPD solid medium containing 300mM LiCl and 10mM H2O2,but there was no significant difference between SN250 and SN250 under other stress conditions.Compared with SN250,the growth of qcr7Δ/Δ was significantly inhibited in YEP solid medium containing non-fermented carbon sources(acetate,ethanol and glycerol),but there was no significant difference in YEP solid medium containing fermented carbon sources(glucose,sucrose and mannitol);5.The experimental results of mycelium growth induction showed that qcr7Δ/Δ grew normally in the liquid medium of YPD+serum and Spider,while the mycelium growth was deficient in the solid medium of YPD+serum and Spider.Compared with SN250,the growth of qcr7Δ/Δ was inhibited in the liquid medium containing different carbon sources,especially in the medium containing non-fermentation carbon sources(glycerol and alcohol),and the growth of qcr7Δ/Δ was completely in yeast state;6.RNA-seq results showed that:(1)compared with qcr7Δ/Δ,a total of 311 genes were obtained in SN250 group,of which 43 genes were up-regulated and 266 genes were down-regulated;(2)through the GO enrichment analysis of differential genes,we found that the differentially expressed genes were mainly concentrated in mycelial hyphal cell wall,cell surface,structural constituent of ribosome,extracellular region,nucleolus and so on.(3)through KO enrichment analysis of differentially expressed genes,we found that differentially expressed genes were mainly enriched in Carbon metabolism,Biosynthesis of amino acids and Ribosome pathways;7.In vitro biofilm experiments showed that QCR7 gene knockout had no effect on the biofilm formation.Conclusions:1.The virulence of Candida albicans can be reduced by the knockout of QCR7 gene;2.Transcriptome analysis showed that the possible regulatory mechanisms of QCR7 gene on Candida albicans mycelium were as follows:(1)affected mycelium growth by regulating energy metabolism pathways such as glucose metabolism,lipid metabolism and amino acid substitution,and(2)participated in mycelial regulation through pathways mediated by RBT1,HWP1,RBT4,RFX2,ZCF5 and ZCF29 genes.