Effect Of The Respiratory Chain Complex Subunit Qcr7 On Pathogenicity Of Candida Albicans And Its Mechanism

Candida albicans is one of the important opportunistic pathogenic fungi.In the body immune function normal condition,the C.albicans and other microorganisms symbiosis in the human body’s mucous membrane surface,generally does not cause the infection.But when the immunity function is low,the C.albicans may cause the human body superficial infection,even endangers the life.Furthermore,the latest warning list of human pathogenic fungi released by the World Health Organization in2022 rated four fungi,including C.albicans,as a critical priority group.Mitochondrial respiratory chain complex III is one of the key structures involved in energy metabolism and plays an indispensable role in the life cycle of eukaryotes.However,little research has been done on the function of mitochondrial respiratory chain complex III in C.albicans and its association with pathogenicity.Objective:To investigate the function of mitochondrial respiratory chain complex III Qcr7subunit in the pathogenic process of C.albicans and the molecular mechanism of biofilm formation.Methods:1.By homologous recombinant gene knockout strategy,Using SN152 as parent strain,the RIP1,COR1,QCR2,QCR6,QCR7,QCR8,QCR9 and QCR10 gene deletion strains were constructed,and named rip1Δ/Δ,cor1Δ/Δ,qcr2Δ/Δ,qcr6Δ/Δ,qcr7Δ/Δ,qcr8Δ/Δ,qcr9Δ/Δand qcr10Δ/Δ.Using qcr7Δ/Δas parent strain,a QCR7gene complement strain was constructed and named QCR7AB.With BCR1,BRG1,TEC1,ROB1,NDT80 and EFG1 gene deletion strains as parental strains respectively,QCR7 gene overexpression strains were constructed.They were named bcr1Δ/Δ+QCR7^OE,brg1Δ/Δ+QCR7^OE,tec1Δ/Δ+QCR7^OE,rob1Δ/Δ+QCR7^OE,ndt80Δ/Δ+QCR7^OE and efg1Δ/Δ+QCR7^OE respectively.Using qcr7Δ/Δas parent strain,HWP1,YWP1,SAP6,XOG1 and HYR1 gene overexpression strains were constructed,named qcr7Δ/Δ+HWP1^OE,qcr7Δ/Δ+YWP1^OE,qcr7Δ/Δ+SAP6^OE,qcr7Δ/Δ+XOG1^OE and qcr7Δ/Δ+HYR1^OE respectively.2.The host systemic infection model was established by tail vein injection in mice.The effect of Qcr7 on the pathogenicity of C.albicans was determined by observing the survival curve,kidney morphology,fungal burden in kidney tissue,pathological degree of kidney tissue and recruitment of immune cells.3.The intracellular ATP content,reactive oxygen species（ROS）level and mitochondrial membrane potential（MMP）of WT,qcr7Δ/Δand QCR7AB strains were detected by luciferase assay,DCFH-DA assay and JC-1 staining,respectively,to evaluate the changes of mitochondrial function.4.A series of phenotypic experiments were conducted to explore the effect of Qcr7 subunit on the pathogenicity of C.albicans and its related molecular mechanism:（1）spot assay were carried out on YPD solid medium with different carbon sources.After 48 h incubation at 30℃,the growth of WT,rip1Δ/Δ,cor1Δ/Δ,qcr2Δ/Δ,qcr6Δ/Δ,qcr7Δ/Δ,qcr8Δ/Δ,qcr9Δ/Δ,qcr10Δ/Δand QCR7AB in different carbon sources were observed.（2）the ability of WT,qcr7Δ/Δand QCR7AB to maintain hyphal growth was observed in Spider solid medium with different carbohydrate as the main carbon source at 37°C for 7 days.（3）the secretion level of aspartic protease was evaluated by observing the hydrolytic loop around WT,qcr7Δ/Δand QCR7AB colonies cultured on BSA solid medium at 37℃for 48 hours.（4）The ability of hyphal polarization growth of WT,qcr7Δ/Δand QCR7AB was detected in Spider liquid medium with different carbohydrates as the main carbon sources.（5）Biofilm formation was induced in 96-well or 12-well polystyrene plates by using Spider liquid medium with various carbohydrates as carbon sources.After incubation at37℃for 48 hours,the biofilm formation of WT,qcr7Δ/Δand QCR7AB under these conditions was analyzed by crystal violet staining.（6）The effects of QCR7 gene overexpression on biofilm formation of bcr1Δ/Δ,brg1Δ/Δ,rob1Δ/Δ,tec1Δ/Δ,ndt80Δ/Δ,efg1Δ/Δ,and the effects of HWP1,YWP1,SAP6,XOG1 and HYR1 gene overexpression on qcr7Δ/Δbiofilm formation were verified in Spider liquid medium with mannitol as the main carbon source.5.Our group previously quantified the WT and qcr7Δ/Δ（OD600=0.8,logarithmic phase of growth）after incubation in RPMI 1640 conditions（simulating in vivo culture conditions）for 4 h.RNA samples were extracted and sequenced by transcriptome.Based on the results of transcriptomic sequencing,the differentially expressed genes between WT and qcr7Δ/Δwere compared in detail,and four kinds of related virulence factor genes after QCR7 gene deletion were identified（hyphal growth,cell adhesion,secretory aspartic protease and biofilm formation）,and its expression was verified by RT-q PCR.At the same time,combined with the expression of QCR7 gene in bcr1Δ/Δ,brg1Δ/Δ,rob1Δ/Δ,tec1Δ/Δ,ndt80Δ/Δand efg1Δ/Δ,the molecular mechanism of biofilm key transcription factors regulating QCR7 gene expression was determined.Results:1.rip1Δ/Δ,cor1Δ/Δ,qcr2Δ/Δ,qcr6Δ/Δ,qcr7Δ/Δ,qcr8Δ/Δ,qcr9Δ/Δ,qcr10Δ/Δ,QCR7AB,bcr1Δ/Δ+QCR7^OE,brg1Δ/Δ+QCR7^OE,tec1Δ/Δ+QCR7^OE,rob1Δ/Δ+QCR7^OE,ndt80Δ/Δ+QCR7^OE,efg1Δ/Δ+QCR7^OE,qcr7Δ/Δ+HWP1^OE,qcr7Δ/Δ+YWP1^OE,qcr7Δ/Δ+SAP6^OE,qcr7Δ/Δ+XOG1^OEand qcr7Δ/Δ+HYR1^OE was successf-ully constructed after homologous recombination gene knockout,nutrient deficiency medium screening and multiple rounds of PCR validation.2.Through the analysis of the carbon source utilization,hyphal growth maintenance and biofilm formation ability of C.albicans complex III subunits,we found that the qcr7Δ/Δstrain had defects in growth on non-glucose carbon sources,especially the inhibition of hyphal growth maintenance and biofilm formation in this mutant appears to be more dramatic than those lacking the other subunits,suggesting that Qcr7 subunit is one of the key subunits in respiratory chain complex III.3.In vivo animal experiments showed that all mice in the qcr7Δ/Δstrain-infected group were still alive on the 21st day,while all the mice in the wild type strain-infected group died within 7 days.The fungal burden in kidney of mice infected with qcr7Δ/Δstrain was significantly lower than that of mice infected with WT strain（P<0.0001）,and the weight of single kidneys and the ratio of both kidney to body weight were also significantly lower than those of mice infected with WT strain（P<0.05）.In addition,the kidneys of mice infected with qcr7Δ/Δstrain were normal in morphology,without tissue injury and inflammatory cell infiltration.The tissue damage and inflammatory cell aggregation were observed in WT infected group,and a large number of hyphal cells were observed by PAS staining.The results showed that the deletion of QCR7 gene could significantly reduce the infection ability of C.albicans to the host.4.In order to further confirm the role of QCR7 gene in carbon metabolism of C.albicans,we compared the growth and mitochondrial function of WT,qcr7Δ/Δand QCR7AB strain under different carbon sources.Spot assay under various carbon sources showed that QCR7 gene deleted plants showed strong growth inhibition in the environment with Glc NAc,maltose,acetic acid,glycerol,acetate and citrate as the main carbon sources.However,there was a certain growth lag on the medium with glucose,mannitol and sucrose as the main carbon sources.The results of mitochondrial function assay showed that the intracellular ATP content of QCR7 gene deletion strain decreased significantly,while the content of reactive oxygen species increased significantly,the level of mitochondrial membrane potential was also lower than that of the wild type.It is suggested that the deletion of QCR7 gene may affect the stability of mitochondrial function of C.albicans.5.The phenotype experiment of virulence factor showed that the hyphal growth maintenance ability of QCR7 gene deleted strains and their hyphal polarization ability in growth inhibition environment decreased,the secretion level of aspartic protease decreased,and the formation of biofilm was limited.It is suggested that the deletion of QCR7 gene can inhibit the function of a series of virulence factors of C.albicans.6.A detailed comparison of the expression levels of genes related to the four virulence factors（hyphal growth,cell adhesion,secretory aspartate protease and biofilm formation）sequenced from the transcriptomes of WT and qcr7Δ/Δstrains showed that many genes related to these four virulence factors were significantly down-regulated,with the largest number of genes（14）and the most significant down-regulation of biofilm-related genes.The results of RT-q PCR confirmed that a series of biofilm-related genes（SAP6,HWP1,XOG1,HYR1,YWP1,PGA7,etc.）were significantly down-regulated after QCR7 gene deletion,suggesting that the reason for the reduced pathogenicity of qcr7Δ/Δstrain may be mainly due to biofilm formation defects.In addition,RT-q PCR analysis further revealed that QCR7 expression was down-regulated in six biofilm core transcription factor deletion strains（bcr1Δ/Δ,brg1Δ/Δ,rob1Δ/Δ,tec1Δ/Δ,ndt80Δ/Δand efg1Δ/Δ）,especially in NDT80 gene deletion strains（P<0.0001）.7.In order to further explore the molecular mechanism of Qcr7 in biofilm formation of C.albicans,we performed overexpression of QCR7 in six biofilm core transcription factor knockout strains,the effect of QCR7 on biofilm formation was evaluated.The results showed that overexpression of QCR7 could partially restore the inhibition of biofilm formation caused by NDT80 gene deletion.Overexpression of HWP1,YWP1,XOG1 and SAP6 in qcr7Δ/Δstrain could partly overcome the biofilm formation defect,and the results were significantly different,but overexpression of HYR1 could aggravate the biofilm formation defect.These results suggest that Qcr7is regulated by Ndt80,and then affects a series of cell surface genes involved in biofilm formation,and finally affects C.albicans biofilm formation.Conclusion:1.Qcr7 is a key subunit of respiratory chain complex III involved in C.albicans pathogenesis;2.Qcr7 is involved in the maintenance of mitochondrial function and has a significant effect on the expression of biofilm related genes in C.albicans;3.The mechanism of Qcr7 in biofilm formation may be regulated by the key transcription factor Ndt80,on the other hand,it regulates the formation of biofilm by affecting the expression of cell surface genes mainly HWP1,YWP1,SAP6 and XOG1.