Candida Albicans SPT20 Regulates Invasion Into Lung Epithelial Cell Through Ras1-cAMP-PKA Pathway

ObjectiveCandida albicans exists widely on the surface of skin and mucosa in most healthy people.It is a common conditional pathogenic fungi clinically.Thanks to the restriction of immune system,C.albicans is in a state of colonization generally and usually causes no symptoms.However,if it changes in the human immunity,physiological function or microbial flora where the fungi colonizes,the state of colonization may be disturbed and infectious diseases like candidiasis may develop.Infectious diseases caused by C.albicans consist of superficial infection and systemic infection.Oral candidiasis,skin candidiasis and vaginal candidiasis are superficial infection.Sepsis caused by C.albicans and infection of internal organs are systemic infection,which may be lethal if serious and medical expenses due to it is high.Mortality of systemic candidiasis remains high even if antifungal agent is used.Many factors contribute to the increasing morbidity of C.albicans infection,including aggressive management of cancer,the increase of HIV infection rate and organ transplants,the extensive use of implantable medical device and overuse of antibiotics.So infection of C.albicans is becoming a global health issue.The pathogenesis of C.albicans is unclear until now.Available antifungal agents are limited in category,narrow in spectrum,obvious side effects and showing up resistance.So treating infection of C.albicans clinically is restricted.Therefore,it is necessary to study the pathogenesis of C.albicans and furthermore explore new targets of curing infection of C.albicans and develop new therapy.C.albicans is pleomorphic and has the morphology yeast,pseudohyphae and hypha.Yeast and hypha play different roles in the development of infection.Invasion into epithelial cells is one of basic virulence factors in C.albicans.There is two different ways to invade into epithelial cells,inducing endocytosis and active penetration.C.albicans induces endocytosis of epithelial cells by binding its invasin to E-cadherin on the surface of epithelial cells.Endocytosis by epithelial cells is a passive process that has no need for reactivity of C.albicans.Two invasins are discovered up to now,Als3 and Ssal.Als3 and Ssal bind to E-cadherin of epithelial cells and induce endocytosis through a clathrin-dependent mechanism.Active penetration is a C.albicans dominating process that needs C.albicans hyphae with activity.Hyphae formation play an important role in this process.It needs the regulation of Rasl-cAMP-PKA signaling pathway to induce endocytosis of epithelial cells in C.albicans.The expression of ALS3 is mediated by transcription factor Efgl,which is at the downstream of Rasl-cAMP-PKA signaling pathway.The function of Efgl can be influenced by PKA.We found in our previous study that the virulence of C.albicans reduced if SPT20 was knocked out.SPT20 plays a part in the formation of hyphae and biofilm in C.albicans.Since SPT20 mediates hyphae formation,it may has an effect on active penetration into epithelial cells.Therefore,the problem is that whether SPT20 mediate the process of inducing endocytosis,what,s the mechanism or pathway if so,whether the regulatory process is invasin and Rasl-cAMP-PKA signaling pathway dependent.In order to solve these problems,we explored the function of SPT20 and the molecular mechanisms by studying the interaction between different strains of C.albicans and A549 cells on the basis of our previous findings.Methods1.Epithelial cells endocytosis assay.C.albicans was cultured in YPD at 30? with 200rpm shaking to get yeast of logarithmic growth phase.A549 cells was cultured in RPMI 1640 with 10%FBS at 37? and 5%CO2.To produce hyphal phase C.albicans,blastospores were incubated in RPMI 1640 medium with 10%FBS in a shaking incubator at 37 ?for 90min.C.albicans hyphae and A549 cells were cocultured for 50min in complete medium at 37? and 5%CO2albicans antibody conjugated with FITC was used to mark adhered C.albicans hyphae outside.Cells and hyphae on slides were rinsed with PBS,fixed with 4%paraformaldehyde and blocked with PBST,which contains BSA and glycine.C.the epithelial cells.After that A549 cells were penetrated with Triton X-100.Calcofluor white was used to mark all the hyphae on the slides,including adhered and endocytosis C.albicans.Observe and count the hyphae using up-right fluorescence microscopy.2.C.albicans damaging epithelial cells assay.C.albicans damaging epithelial cells was tested using a cytotoxicity LDH test kit.The amount of dead and damaged epithelial cells was evaluated by test the LDH concentration of culture supernatants.At first,Pre-experiment should be perform to determine the suitable amount of A549 cells.Then the formal experiment was done.Different strains of C.albicans and A549 cells were cultured.Both were counted and cocultured 20h in complete medium.Culture supernatants were transferred to 96-well plates and reagents in the kit were added according to the manual.OD490nm of supernatants was determined with microplate reader to evaluate the relative density of LDH.3.Real time PCR.Total RNA isolation system was used to isolate total RNA of different C.albicans strains.C.albicans was cultured overnight to obtain yeast at logarithmic growth phase and counted.Cell wall of C.albicans was broken with lyticase.Then total RNA was isolated according to the manual of the kit.The density and purity of isolated RNA were determined using UV spectrophotometer.A reverse transcription kit was used to remove genomic DNA in total RNA and perform the reverse transcription reaction using total RNA as template to obtain cDNA according to the system and program in the manual.Removing the genomic DNA is necessary as genomic DNA in total RNA may interfere with rt-PCR.SYBR Green I rt-PCR was conducted using an rt-PCR kit with cDNA of different C.albicans strains as templates.After rt-PCR reaction,relative expression of related genes in different strains was analyzed by 2-??Ct method.4.PKA activity assay.PKA activity of different C.albicans strains was determined with a PKA activity kit,which employed a fluorescent peptide as substrate,and the peptide had high specificity to PKA.The substrate was phosphorylated in test and its net charge changed from +l to-1,which made phosphorylated substrate and unphosphorylated substrate easy to be separated when electrophoresis began.PKA activity of different samples was evaluated with the fluorescence intension of phosphorylated substrate.C.albicans was cultured overnight to obtain yeast at logarithmic growth phase and counted.1 ×108 yeast cells of different strains were centrifugalized and cell wall of the yeast was broken by glass beads method.Samples were prepared according to the manual of the kit.Test group,PKA positive control group and PKA negative control group were set up.Agarose gel electrophoresis was performed to separate the phosphorylated substrate and unphosphorylated substrate peptide in the samples.Photos of the gel after electrophoresis were taken using a GEL imaging system.Results1.The efficiency to induce endocytosis of A549 cells decreased in C.albicans with SPT20 knocked out.In order to clarify whether SPT20 is related with the process C.albicans inducing the endocytosis of epithelial cells,we conducted the epithelial cells endocytosis assay.The strains of WT,spt20?/? and spt20?/SPT20 were cocultured with A549 cells respectively and the strains interacted with epithelial cells.Observe the interaction between the three strains and epithelial cells with a microscope after coculture.In order to mark C.albicans and distinguish adherence from endocytosis,at first C.albicans antibody conjugated with FITC was used to mark the C.albicans outside the epithelial cells,which presented green.Then A549 cells were penetrated with Triton X-100.After that calcofluor white was used to mark all the hyphae on the slides,including adhered and endocytosis C.albicans,which presented blue.Blue C.albicans minus green C.albicans is the amount of endocytosis.The experiment was triplicated separately.At least 100 C.albicans cells were counted each slide.The result showed that endocytosis of sP120?/? was significantly less than that of WT.Therefore,compared with WT,the efficiency to induce endocytosis of epithelial cells decreased significantly in spt20?/? strain.2.The efficiency to damage A549 cells decreased in C.albicans with SPT20 knocked out.In order to clarify whether SPT20 is related with the process C.albicans damaging A549 cells,we conducted the C.albicans damaging epithelial cells assay to explore the role SPT20 plays in damaging A549 cells.At first C.albicans strains of WT,sPt20?/? and spt20?/SPT20 were cocultured with A549 cells.Then the density of LDH in the culture supernatants was determined with a LDH kit to evaluate the extent of damage.The experiment was triplicated separately and each time a sample was repeated three holes as technical repeats.OD490 of culture supernatants was used to e'valuate the relative density of LDH.It turned out that the LDH density of spt20?/? and A549 cells cocultured supernatants was lowest.Therefore,compared with WT,the efficiency to damage A549 cells decreased significantly in spt20?/? strain.3.The activity of PKA decreased in C.albicans with SPT20 knocked out.Samples were prepared by breaking cell wall of different C.albicans strains using glass beads method.Then the PKA activity of WT,spt20?/? and spt20?/SPT20 strains was determined using a PKA activity kit to explore whether SPT20 influences the activity of PKA in C.albicans.It showed that compared with WT and spt20?/SPT20 strains,PKA activity of spt20?/? strain decreased significantly.Therefore,the activity of PKA decreased in C.albicans with SPT20 knocked out.SPT20 play a part in the activity of PKA in C.albicans,4.Expression of ALS3 and RAS1redueed in C.albicans with SPT20 knocked out.Total RNA was isolated from WT,sPt20?/? and spt20?/SPT20 strains.Reverse transcription was conducted using total RNA as template to obtain cDNA.Relative expression of ALS3 and RAS1 in the three strains was detected by rt-PCR using cDNA as template.The experiment was triplicated separately and each time a sample was repeated three holes as technical repeats.The result showed that compared with WT and spt20?/SPT20 strains,expression of ALS3 and RASI reduced significantly in spt20?/? strain.ConclusionC.albicans SPT20 mediates the expression of RASI.RAS1 regulates the activity of PKA through Rasl-cAMP-PKA pathway.Function of transcription factor Efgl is influenced by PKA.Efgl plays a role in the expression of invasin gene ALS3.The invasin Als3 mediated the process C.albicans inducing the endocytosis of epithelial cells and invading into the epithelial cells.In a word,it is our conclusion that C.albicans SPT20 regulates invasion into lung epithelial cell through Rasl-cAMP-PKA pathway.