Antimicrobial Spectrum And Mechanism Of Clioquinol In Vitro

Objective:Fungal and bacterial infections are common clinical diseases.In recent years,the increase of immunosuppressive population,the increase of drug-resistant strains and the limitations of antimicrobial drugs make the treatment more and more challenging.Therefore,new antimicrobial drugs are urgently needed to treat infectious diseases.Clioquinol(5-chloro-7-iodo-8-hydroxyquinoline)could inhibit growth of some bacteria and fungi.The study aimed to investigate the antimicrobial spectrum and activities of clioquinol against common pathogenic bacteria and fungi in vitro,and to explore the antimicrobial mechanism of clioquinol.Trying to provide evidences for its clinical application.Materials and Methods:1.Antimicrobial spectrum and activities of clioquinol in vitro.①Evaluate the antimicrobial spectrum and activities of clioquinol bulk drug and 3%clioquinol cream in vitro,and compared with other antimicrobial drugs;②Measure the minimum fungicidal concentration of clioquinol and evaluate the fungicidal function of clioquinol through time-kill curve;③ Observe morphology changes of yeast treated with clioquinol under fluorescence microscope and scanning electron microscope.2.Analyse differential gene expression profile of Candida albicans treated with clioquinol through transcriptome sequencing.Transcriptome sequencing revealed the possible metabolic pathways that clioquinol may affect C.albicans,and verify the gene expression and protein expression of related pathways.3.Clioquinol affects energy metabolism and induces apoptosis.① Evaluate the adenosine triphosphate level of C.albicans cells treated with clioquinol;②Observe the early and late apoptosis phenomenon of C.albicans cells treated with clioquinol;③ Evaluate the reactive oxygen species and calcium ion levels changes in C.albicans cells treated with clioquinol.Then measure mitochondrial membrane potential,cytochrome C levels and metacaspase activities of C.albicans cells treated with clioquinol to investigate the antimicrobial mechanism of clioquinol.4.The effect of clioquinol on cell wall,cell membrane,virulence factors and ion homeostasis of C.albicans.① Measure the changes of genes expression and enzyme activities of glucanase,glucan synthase,squalene epoxidase and lanosterol demethylase of C.albicans cells treated with clioquinol;② Measure major contents of cell wall(mannose,chitin and glucan)of C.albicans cells treated with clioquinol and sorbitol protection assay was used to evaluate the effect on C.albicans cell wall;③ Cell membrane integrity and potential were evaluated in cells treated with clioquinol,and ergosterol binding assay was used to evaluate the effect of clioquinol on C.albicans cell membrane;④Observe the effect of clioquinol on yeast-hyphae transformation in multiple liquid and solid media,and evaluate the effect of clioquinol on biofilm through qualitative and semi-quantitative assay.Evaluate the cell surface hydrophobicity of C.albicans cells treated with clioquinol and the effect of clioquinol on host immune recognition through β-glucan exposure assay;⑤ Assess the effect of exogenous metal ions on antifungal activity of clioquinol on C.albicans,measure the minimum inhibitory concentration of clioquinol against C.albicans in media with exogenous metal ions or chelators,and measure the ferrous ion level of cells treated with clioquinol.Results:1.① Clioquinol could inhibit the growth of Candida spp,Malassezia spp,Dermatophytes,Sporothrix globosa,Aspergillius terreus and Fusarium solani,but it had no effect on growth of Escherichia coli,Staphylococcus aureus,Staphylococcus epidermidis and Propionibacterium acnes;② Clioquinol had fungicidal function against C.albicans in time and concentration dependent manner;③ There were obvious morphology changes of Candida spp and Malassezia furfur cells treated with clioquinol,including depression of spores surface,decrease of hyphae formation and budded spores rate.2.① The differentially expressed genes of C.albicans cells treated with clioquinol mainly focused on the energy metabolism pathways;②Clioquinol could down regulate the gene expression and enzyme activities of C.albicans of tricarboxylic acid cycle and oxidative phosphorylation pathways,and lead to the decrease of final product-adenosine triphosphate;③Clioquinol could down regulate the gene expression and enzyme activities of catalase and superoxide dismutase in antioxidant system of C.albicans.3.① Clioquinol could decrease the adenosine triphosphate levels in C.albicans cells in concentration-dependent manner;②Clioquinol could significantly increase both reactive oxygen species and calcium ion levels in C.albicans cells.Then clioquinol could induce mitochondrial membrane potential depolarization and cytochrome c release,and finally activate the metacaspase and induce cell apoptosis.Typical apoptotic characteristics(phosphatidylserine externalization,DNA and nuclear fragmentation)could be observed in C.albicans cells treated with clioquinol;③N-acetylcysteine(reactive oxygen species scavenger)could weaken or reverse these changes induced by clioquinol,and ultimately reversed the apoptosis C.albicans cells induced by clioquinol.4.① Clioquinol could up regulate the genes expression and enzymes activities of glucanase and glucan synthase,but down regulate the genes expression and enzymes activities of squalene epoxidase,and had no obvious effect on genes expression and enzymes activities of lanosterol demethylase;②Sorbitol assay demonstrated that clioquinol had no effect on cell wall.Clioquinol did not affect the mannose,chitin and glucan levels either;③ Ergosterol binding assay demonstrated that clioquinol could not inhibit the fungal growth through binding to the ergosterol in cell membrane.Low concentration clioquinol could influence cell membrane potential while no obvious effect was observed in cell membrane integrity.High concentration clioquinol could influence cell membrane potential and integrity;④Clioquinol could inhibit hyphae formation in multiple liquid and solid media and inhibit biofilm formation in time and concentration-dependent manner.It also decreased cell surface hydrophobicity and promoted C.albicans cells recognized by host through β-glucan exposure in concentration-dependent manner;⑤ Antifungal activity of clioquinol could by reversed by metal ions.Metal ions could increase the minimum inhibitory concentration of clioquinol while metal chelators could decrease the minimum inhibitory concentration of clioquinol.The intracellular ferrous irons decreased with the increase of clioquinol concentration.Conclusion:1.Clioquinol could inhibit the growth of most common pathogenic fungi.Clioquinol had no obvious effect on growth of bacteria.2.Clioquinol could affect the energy metabolism and antioxidant system of C.albicans.3.Clioquinol could induce mitochondrial dysfunction through up regulating reactive oxygen species and calcium ions levels in C.albicans cells,then induce apoptosis in metacaspase-dependent manner.4.Clioquinol could influence the activities of glucan synthase and glucanase of C.albicans,but had no obvious effect on cell wall major contents.5.Clioquinol had effect on squalene epoxidase and could affect cell membrane potential and integrity of C.albicans.6.Clioquinol could decrease the virulence factors of C.albicans through inhibiting yeast-hyphae transformation and biofilm formation,decreasing cell membrane hydrophobicity and promoting host immune recognition.7.Clioquinol could inhibit fungus growth through affecting metal ions homeostasis in C.albicans cells by chelating copper,iron and zinc ions.