Research Of Interactions Between Malassezia Furfur And Staphylococcus Epidermidis

Background: Malassezia is a genus of yeasts that residents on the skin surface of human and warm-blooded animals and sometimes acts as an opportunistic pathogen.Under the conditions of predisposing factors,such as lower host defense and changes in skin microenvironment,Malassezia could cause tinea pityriasis and Malassezia folliculitis and be associated with the occurrence of skin disease,including seborrheic dermatitis,dandruff,atopic dermatitis and psoriasis.Seborrheic dermatitis(SD)and dandruff(D)are relapsing skin diseases with erythema and scaling.Dandruff was usually considered as a mild non-inflammatory form of SD.Although the pathogenesis of SD/D were still unclear,various internal factors and external environmental factors had been reported to be related to the occurrence and development of SD/D,such as microorganism colonization,serum status,immune state,skin barrier function and individual susceptibility.It was proved in the research of antifungal drug reactions that the genus of Malassezia was associated with SD/D.Researches demonstrated that partial application of antifungal drugs could reduce Malassezia colonization on the skin lesion and alleviate symptoms of SD/D.Malassezia could interplay with keratinocytes and immune cells to induce a nonspecific immune response.Combination of Toll-like receptor 2(TLR2),Malassezia could promote the production of IL-8 from keratinocytes,which participated in the development of SD.Of all Malassezia species,Malassezia globosa(M.globosa),Malassezia restricta(M.restricta)and Malassezia furfur(M.furfur)are the most commonly isolated species from the lesions of SD/D at significantly high rates in comparison with the normal subjects.Besides the disequilibrated colonization of Malassezia on the lesions,an increasing colonization of Staphylococcus epidermidis(S.epidermidis)were also observed in SD/D.Being the prominent part of skin microbiota,the changes in quantity or proportion of S.epidermidis would lead to a disequilibrium of other flora,which might relate to the occurrence of skin disease.Also,the formation of S.epidermidis biofilm was reported to be involved in the pathogenesis of skin diseases.Recent studies found that lipoteichoic acid(LTA)produced by S.epidermidis played anti-inflammation roles in multiple skin diseases,but it was still uncertain that the same anti-inflammation effect could occur in skin disease induced by Malassezia.However,in SD/D conditions,Malassezia and S.epidermidis both showed disequilibrium on skin lesions.We speculated there might be interactions between these two microorganisms which led to an imbalanced microbiota colonization of the skin.Through our former research,the supernatant of M.furfur(SMF)promoted the growth of S.epidermidis.Based on the previous researches,several questions below would be prepared to investigate: What were the effects of SMF on the biofilm formation of S.epidermidis? What were the effects of SMF on protein expression of S.epidermidis? What would it take for SMF to affect the growth and biofilm formation of S.epidermidis? What kind of cytokines would keratinocytes produce after being stimulated by M.furfur and what was the difference if adding LTA?To answer the questions above,our study started with the interactions between Malassezia and S.epidermidis.The promotion of SMF on growth of S.epidermidis was determined by quantitative method.The semi-quantitative crystal violet staining method and confocal microscopy were used to study the biofilm formation.The protein components of SMF and the differential expression proteins of S.epidermidis influenced by SMF were performed by proteomic analysis.The mechanisms behind growth and biofilm formation of S.epidermidis affected by SMF were explored and verified.Real-time PCR,ELISA and Western blot were used to observe the production of cytokines and the expression of proteins in NF-?B signal pathway induced by Malassezia with LTA.Material and Methods: 1.The experimental objects: M.furfur(ATCC?14521?)and S.epidermidis(ATCC?12228?)were purchased from American Type Culture Collection(ATCC).S.epidermidis(ATCC?35984?)were kindly provided by professor Qu di(Key Laboratory of Medical Molecular Virology of Ministries of Education and Health,Shanghai Medical College of Fudan University,Shanghai,China).Immortalized human keratinocyte,Ha Ca T cell line was used in this study.2.Preparation of SMF: M.furfur was adjusted to 1.0�107 colony forming units (CFUs)/m L with Beef extract-sodium chloride-peptone(BSCP)and was put into 30? with shaking overnight(15h).After centrifugation and filtration,the supernatant was collected as SMF for further study.3.S.epidermidis growth measurement: S.epidermidis were measured by testing the bacteria turbidity hourly at 600 nm by microplate reader.The growth curve of each group was mapped according to the optical density(OD)values of bacteria at different hours at 37?.Another plate was prepared as mentioned above at the same condition for 12 hours.The bacteria suspension was removed to a new tube and the number of the bacteria in each sample was counted by the flow cytometer.4.S.epidermidis(ATCC?35984?)biofilm formation: The semi-quantitative crystal violet staining method and confocal microscopy were used to study the biofilm formation.Absorbance was read at 570 nm in microplate reader.The sterile coverslips were examined by confocal laser scanning microscopy and fluorescence microscope.5.Proteome analysis of M.furfur and S.epidermidis(ATCC?12228?): Mass spectrometry and bioinformatics analysis method were used to resolve proteins in SMF and explore the differentially expressed proteins in S.epidermidis which were influenced by SMF.6.Detection the expression of target proteins by parallel reaction monitoring(PRM)and Real-time PCR: The differently expressed proteins(DEPs)were confirmed by PRM analysis.Proteins in SMF were extracted and then digested by trypsin.Peptide fragment was analyzed by liquid chromatography-mass spectrum(LC-MS)and data were processed by quantitative analysis.The expression of m RNA was detected by Real-time PCR.Total RNA was extracted by RNA prep Pure Bacteria kit.c DNA was synthesized from RNA by Fast Quant RT Kit.RT-Q PCR was performed with Super Real Pre Mix Plus kit.Ct values were calculated by relative quantification.7.Measurement of p H value,NH4+,urease and urease activity: The p H value of SMF and BSCP were measured by p H reagent.We used phenol-sodium hypochlorite method to test the NH4+ level in SMF and BSCP.The OD values at a wavelength of 640 nm were used to calculate the urea level.The urease of M.furfur was detected by urease identification medium.The urease activity assay kit was used to test the urease activity of M.furfur in different conditions.The absorbance was measured at a wavelength at 670 nm and the OD values were proportionated to the urease activity.8.Morphological observation by Transmission electron microscopy(TEM): M.furfur was fixed,dehydrated,embedded and sectioned.The sections were stained and then viewed by transmission electron microscopy.9.Measurements of cytokines by Real-time PCR and ELISA: Real-time PCR and ELISA were used to evaluate the effects of Malassezia on the production of cytokines.Operating steps of PCR were the same as above.The procedures of ELISA included adding samples,washing plates,incubating antibodies,washing plates and determining the OD values.10.Analyze the expression of NF-?B pathway related proteins by western blot: The expression of P65,p-P65,I?B? and p-I?B? were detected by western blot.Total protein was extracted,and the concentration was measured with BCA method.Operation sequence included running the gel,transfering patterns,sealing and incubating antibodies.The bands were visualized with ECL Western Blotting Substrate.11.Statistical analysis: All experiments were repeated at least three times and the results were presented as mean�SEM.The figures were generated using Graph Pad 6.0 software.For experiments with only two groups,unpaired t test was performed for comparison.For more than two groups of experiments,the differences between each group were analyzed by one-way analysis of variance(ANOVA).The results were considered statistically significant only if p<0.05.Results: 1.Effects of SMF on the growth and the biofilm formation of S.epidermidis.The growth of S.epidermidis was promoted by SMF compared with the BSCP control group.The promoting effect of SMF showed a concentration-dependent manner.Similar results were obtained when counting bacteria by flow cytometry.Compared with the BSCP group,undiluted SMF group and the 5-fold diluted SMF showed higher promoting effect(p<0.05).The biofilm formation of S.epidermidis was increased after the treatment of SMF.The confocal laser scanning microscopy images confirmed the promoting effect of SMF on biofilm formation.Confocal microscopy detected a dense membrane like biofilm structure in SMF group,while the control group showed a loose biofilm structure.2.Proteins of SMF: Protein analysis of SMF detected 102 proteins.Among these,one third were uncharacterized proteins presumed to be existed,one third were genomic scaffold protein and the rest were proteins with specified function,such as protease,lipase,phospholipase and so on.3.Effects of SMF on proteome of S.epidermidis(ATCC?12228?): proteomic analysis showed that 113 proteins with 54 upregulated and 59 downregulated were significantly affected by SMF with the fold change cut-off being 1.3.Differentially expressed proteins involved in virulence were downregulated.Arginine biosynthesis pathway was enriched on proteome analysis.We detected four proteins involved in arginine biosynthesis.Three of them were major enzymes involved in catalyze Arginine to ornithine,ammonia and carbon dioxide.Ammonia played a vital role in the survival of S.epidermidis under skin acid stress.The expression of related enzymes participated in decomposition of arginine decreased in our study,which indicated that the p H value of SMF was higher than that of BSCP.4.Verification of differently expressed proteins by PRM and Real-time PCR: The expression trends of target proteins verified by PRM were in accordance with those in proteomics.The m RNA expression of 4 proteins in arginine biosynthetic pathway was verified by PCR,and 3 of them presented lower trend,which showed the same trend with results of proteomics.5.The elevated p H value in SMF promoted the the growth and the biofilm formation of S.epidermidis: The p H level of SMF was higher than BSCP.We compared the growth of S.epidermidis and biofilm formation in the absence of SMF at p H5 and p H6 value and found the growth of S.epidermidis was promoted at p H6 value.Adjusting the p H value of BSCP to the same as SMF,we observed no difference in the growth S.epidermidis.6.Urease increased p H value in SMF: Urease produced by M.furfur was detected by urease identification medium.Urease catalyzed hydrolysis of urea into ammonia and led to the increase of p H value in the culture medium.We co-cultured AHA with M.furfur and then extracted SMF,of which the p H value only showed minor increase.Therefore,it was believed that M.furfur increased p H value by urease.7.The effect of lipid conditions on the morphology and urease activity of M.furfur: Cultured in lipid-free condition,the cell wall of M.furfur was slightly thickened and decreased in permeability.More filaments were seen in the outer wall,while the jagged structure of the inner wall disappeared and became smoothed.Part of the cell walls were damaged.Heteromorphism and necrosis were seen in the organelles.Increased urease activity of M.furfur was detected after cultured in lipid-free condition(m Dixon without lipid components)compared with the normal culture medium in m Dixon,suggesting the urease activity of M.furfur was increased without lipid.8.Staphylococcal LTA inhibited the production of cytokines in Ha Ca T cells induced by M.furfur: M.furfur induced IL-6 and IL-1? m RNA expression in Ha Ca T cells after co-cultured for 1h.LTA inhibited the m RNA expression of IL-6 and IL-1? induced by M.furfur.ELISA measurements observed the similar trend of IL-6 in cell cultural supernatant.9.Staphylococcal LTA inhibited the production of cytokines through NF-?B signal pathway: Co-culture with M.furfur for 30 min,the expression of p-P65 in Ha Ca T cells was increased.While p-P65 expression was decreased after pretreatment with LTA.Conclusion: 1.SMF promoted the growth and biofilm formation of S.epidermidis.2.Urease activity existed in M.furfur,which could catalyze hydrolysis of urea into ammonia and increase the p H value in the culture medium.The changing p H values of cultivation environment(approximately from p H5.0 to p H6.0)provided favorable condition for the growth and biofilm formation of S.epidermidis.The increasing quantity of S.epidermidis might cause disequilibrium in skin flora.While the increasing biofilm formation of S.epidermidis might obstruct sweat gland and sebaceous gland,which took part in pathogenesis of skin disease.3.The morphology of M.furfur made a difference and urease activity increased after cultivation in lipid-free condition.4.Induced by M.furfur,the m RNA expression of IL-6 and IL-1? in Ha Ca T cells was elevated and secretion of IL-6 in cell cultured supernatant was increased.While Staphylococcal LTA could inhibit the production of cytokines induced by M.furfur.M.furfur could promote phosphorylation of P65 protein in Ha Ca T cells and then induce the expression of downstream cytokines.LTA could inhibit the phosphorylation of P65 protein,thus inhibiting the production of cytokines induced by M.furfur.