Morphology, Cell Wall Polysaccharide Structure And Its Immunological Effect Of Candida Albicans

Chapterâ… Preliminary mechanism study of polymorphic Candida albicansSectionâ… Effect of amino acid on filamentation of Candida albicansObjective To investigate the effect of amino acid on the filamentation of Candida albicans.Methods A basal synthetic culture medium was established with references to synthetic medium SD.Synthetic medium SD consists of yeast nitrogen base with 2% glucose but without amino acids.One of the 17 amino acids at 10mmol/L concentration was added to synthetic medium SD as a single component of amino acid,and the morphology of colony and cell of Candida albicans was examined.The morphology of Candida albicans was examined under anaerobic condition with or without glucose. Results L-arginine induced filament.L-cysteine,L-threonine,L-valine and L-tryptophan induced perfect yeast cells.Mixture of filament and yeast could be detected when L-alanine,L-aspartic acid,L-phenylalanine,L-glycine,L-histidine,L-isoleucine, L-lysine,L-leucine,L-methionine,L-asparagine,L-proline,and L-tyrosine were used as a single amino acid.All the colonies were smooth on SD plate with or without single amino acid,and L-arginine resulted in colonies with small spinules.Candida albicans growth was inhibited and only yeast could be detected after incubation with or without glucose under anaerobic condition.Conclusions Filament of Candida albicans could be induced by L-arginine.No filament of Candida albicans could be induced by L-arginine under anaerobic condition.Sectionâ…¡Effect of pH and oxygen on the filamentation of Candida albicansObjective To investigate the effect of pH and oxygen on the filamentation of Candida albicans.Methods Candida albicans was incubated in Muller-Hinton broth at 37℃for induction of filamentation in vitro.Growth curve,doubling time and percent of filamentation of Candida albicans were observed under aerobic,anaerobic condition and different pH value at intervals.Results Candida albicans growth was slow and no hyphae but yeast could be found under anaerobic condition.No difference of growth rate in lag phase of Candida albicans under different pH value condition was found.Growth rate of Candida albicans in log phase was slower under pH3,pH4 than pH5,pH6,pH7, pH8 and pH9,Filamentation percent of Candida albicans was less than 20%under pH3, pH4 and pH5.Filamentation percent of Candida albicans was up to 70%under pH6, pH7,pH8 and pH9.Conclusions Hyphae was completely inhibited and only yeast form could be detected while Candida albicans were incubated under anaerobic condition. Candida albicans can thrive at a wide range of extracellular pH from 3 to 9.Acidic conditions favor yeast growth,while alkaline conditions favor hyphal growth.Sectionâ…¢Effect of electron transfer system on the hyphal formation of Candida albicansObjective To investigate the effect of electron transfer system on the hyphal formation of Candida albicans.Methods Candida albicans was incubated in RMPI1640 supplemented with 10%newborn calf serum at 5%CO₂ 37℃for induction of hyphal formation in vitro.Growth curve,morphology and percent of filamentation of Candida albicans were observed with or without inhibitors or activator of electron transfer system at intervals.MTT assay was used to assess the viability of Candida albicans.Results No difference of growth and filamentation of Candida albicans,could be found between solvents(chloroform and dimethyl sulfoxide) and control groups.Only yeast cell of Candida albicans could be detected after incubation with thenoyltrifluoroacetone(TTFA) or benzhydroxamic acid for 24 hours.Growth of Candida albicans were significantly inhibited in log phase when adding classic respiratory chain inhibitors such as rotenone, antimycin A,oligomycin,sodium azide,TTFA and malonic acid(P＜0.01). Benzhydroxamic acid significantly inhibited growth of Candida albicans in log phase (P＜0.01).When Candida albicans were incubated with rotenone,antimycin A, oligomycin,sodium azide,TIFA,malonic acid,benzhydroxamic acid and GMP, filamentous percent of Candida albicans at 12h was 87.49±0.52,48.75±4.44,50.33±8.50, 99.00±1.00,1.60±0.53,94.01±0.99,0.00±0.00 and 92.33±2.08,and inhibition percent of Candida albicans at 7h was 1.34±0.15,70.61±1.02,50.63±5.38,17.80±7.89,45.17±1.27, 10.75±3.62,72.46±1.14 and -(5.96±4.07),respectively.When Candida albicans were incubated with rotenone,antimycin A,oligomycin,benzhydroxamic acid and TTFA,the ratio of GSSG/GSH were significantly higher than the control(P＜0.05).Conclusions Hyphal formation of Candida albicans is suppressed by inhibitors of both electron transfer systems.Hyphal formation is mainly controlled by AOX pathway.Inhibition of AOX pathway leads to oxidative stress and suppression of hyphal formation.Sectionâ…£Effect of temperature and carbon dioxide on the white opaque switch of Candida albicans and pathogenic difference study of the white and opaque form of Candida albicansObjective To investigate the effect of temperature and carbon dioxide on the white opaque switch of Candida albicans and pathogenic difference study of the white and opaque form of Candida albicans.Methods Modified Lee's medium supplemented with phloxine B was used to study phenotype switch under different temperature and concentration of carbon dioxide.White and opaque Candida albicans and THP-1 were co cultured,and the intracellular and extracellular kill activity was studied.Results Proportions of opaque colony under 0.03%CO₂ 25℃,0.03%CO₂ 37℃and 5%CO₂37℃were 0.572±0.087,0.920±0.030 and 0.985±0.026,respectively,when evaluating white to opaque switching.Proportions of opaque colony under 0.03%CO₂ 25℃,0.03%CO₂37℃and 5%CO₂37℃were 0.60±0.114,0.983±0.003 and 0.998±0.003,respectively,when evaluating opaque to white switching.THP-1 mediated extracellular white and opaque form of Candida albicans killing activity was 62.98%±5.02%and 87.07%±1.80%, respectively.There was significantly difference of THP-1 mediated intracellular Candida albicans killing activity between white and opaque form(P＜0.05).Conclusions CO2 and 37℃stimulate white to opaque switching.CO₂ and 37℃block opaque to white switching.THP-1 mediated Candida albicans killing activity is higher in opaque form than that in white form.Chapterâ…¡Isolation,purification,physiochemical and structural analysis of mannan and insoluble glucan from cell wall of Candida albicans hyphal and yeast formSectionâ… Isolation,purification,physiochemical and structural analysis of mannan from cell wall of Candida albicans hyphal and yeast formObjective To analysis physiochemical and structural information of mannan from cell wall of Candida albicans hyphal and yeast form.Methods Hyphal and yeast form of Candida albicans were induced in vitro.Alkali-extracted mannan was deproteinated, dialyzed,concentrated,centrifuged and lyophilized,then was further purified by using a DEAE-cellulose column.The eluate was pooled according to the detection by modified phenol-sulfuric acid analysis.Structures were elucidated using monosaccharide composition analysis by gas chromatography(GC),methylation analysis by gas chromatography-mass spectrometry(GC-MS) and nuclear magnetic resonance(NMR) spectroscopy.Results Mannan from cell wall of yeast and hyphal form both showed a single symmetrical peak on high-performance gel-permeation chromatography (HPGPC),and their molecular weight were 30kd and 23kd,respectively.1,6-linked, 1,2-linked,1,3 linked and non-terminal mannan residues were found,a conformation dominates,whileβconformation was still found.Conclusions.It showed that mannan from both yeast and hyphal form contained 1,6-linked backbone with a side chain mainly containing 1,2,1,3 linked mannan residues,whereas yeast form contained a longer 1,2 -linked mannose branch.Sectionâ…¡Isolation,purification,and structural analysis of mannan and insoluble glucan from cell wall of Candida albicans hyphal and yeast formObjective To isolate and analysis structure information of insoluble glucan from cell wall of Candida albicans hyphal and yeast form.Methods.Hyphal and yeast form of Candida albicans were induced in vitro.Alkali-acid method was used for isolation. Sulfated method was used to soluble the insoluble glucan.Structures were elucidated using monosaccharide composition analysis by gas chromatography(GC),methylation analysis by gas chromatography-mass spectrometry(GC-MS) and nuclear magnetic resonance(NMR) spectroscopy.Results Insoluble glucan was isolated from cell wall of both yeast and hyphal forms.1,3 linked glucan residues was found by methylation analysis.Onlyβconformation was found by ¹³C NMR spectroscopy Conclusions It showed that both of insoluble glucan from yeast and hyphal form containedβ,1,3-linked linear glucan,and no difference was found in composition,link style and conformation.Chapterâ…¢Immune response of fungal cell wall polysaccharide on the monocyte-macrophage cell line THP-1Objective To investigate the immune response of monocyte-macrophage cell line THP-1 to fungal cell wall polysaccharide.Methods Semi-quantitative RT-PCR was used to study the mRNA expression of Dectin-1 and TNF-a.ELISA was used to measure the cell culture supernatant.Flow cytometry was used to detect the Dectin-1 expression on the cell membrane.Results Dectin-1 and TNF-a mRNA were expressed in monocyte-macrophage cell line THP-1 when treating with various dose Zymosan at certain intervals.THP-1 cell secreted TNF-a in dose-dependent and time dependent way when treating with heat-inactivated hyphae,heat-inactivated yeast,Zymosan,hyphal mannan,yeast mannan and insoluble glucan.THP-1 cell membrane Dectin-1 protein expression was found in time-dependent way when treating with Zymosan and insoluble glucan.Conclusions.TNF-a secretion of THP-1 cell was higher when treating with heat-inactivated Candida albicans yeast than heat-inactivated Candida albicans hyphae. TNF-a secretion of THP-1 cell was higher when treating with Candida albicans cell wall mannan in yeast form than in hyphal form.Dectin-1 mediated TNF-a secretion of THP-1 cell when treating with Zymosan and insoluble glucan.