| Background:Trichosporon species are considered an important pathogen of invasive infections,with high mortality and poor prognosis.As the last defense for invasive Trichosporon infections,the development of azoles resistance is alarming.In the present study,we aimed to investigate the molecular epidemiology and virulence distribution characteristics of invasive Trichosporon infections in China,as well as the mechanism of azoles resistance.Methods:A total of 133 non-duplicated Trichosporon isolates were collected from 43 medical centers which participate the CHIF-NET program during 7-year period,from August,2009 to July,2016.Accurate identification was achieved by sequencing analysis.In vitro antifungal susceptibilities were performed using the Clinical and Laboratory Standards Institute(CLSI)broth microdilution method.Tentative epidemiologic cutoff values(ECVs)derivatives for T.asahii isolates were established.The identification performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)and detection performance of Sensititre YeastOne plates were evaluated.Based on in vitro experiments,we investigated the extracellular enzyme activity distribution and biofilm formation ability,structure and drug resistance for Trichosporon isolates.For the multiple azoles highly resistant clinical isolate of T.asahii,we explored the efflux pump activity,sequencing and expression analysis of the genes related to azole resistance,and transcriptome sequencing analysis.Results:A total of 10 species were identified in the multicenter invasive Trichosporon infections in China.T.asahii remains the leading pathogen(81.2%),and blood is the most common type of specimen(36.8%).By IGS sequencing,five kind of genotype were identified in T.asahii.Genotype 1(41.7%),genotype 4(31.5%)and genotype 3(23.1%)are the main genotypes in China.The overall distribution of MIC values of T.asahii to amphotericin B was higher than that of non-asahii Trichosporon isolates,with GM values of 1.36 ?g/ml and 0.7 ?g/ml,respectively.47.2%of T.asahii(51/108 isolates)have MIC values?2 ?g/ml against amphotericin B,while non-asahii Trichosporon isolates(25 isolates)have MIC values<1 ?g/ml.High fluconazole MICs(?8 ?g/ml)were observed for 25%of T.asahii(27/108 isolates),2 isolates of T.dohaense and each one isolate of T.asteroides,T.mucoides and T.faecale.Voriconazole was the most potent antifungal agent in vitro,with a GM of 0.09 ?g/ml.The tentative ECV values for fluconazole,voriconazole,itraconazole and posaconazole were 16?g/ml,0.25?g/ml,1 ?g/ml and 1 ?g/ml,respectively.While the ECV values for amphotericin B and 5-flucytosine were 4 ?g/ml.Overall,Bruker Biotyper showed better identification performance than Vitek MS for non-asahii Trichosporon isolates,with correct identification rate of 52%versus 32%(P=0.152).The Sensititre YeastOne plates showed high consistency with broth microdilution method.At least 70.0%of Trichosporon isolates have DNase,hemolysin and esterase activities,while the proportion that produce phospholipase,casein and gelatinase are relatively low,with 54.1%,5.3%and 59.4%,respectively.For T.asahii isolates,genotype 3 has stronger ability to produce hemolysin,genotype 4 has stronger ability to produce esterase,and all the 3 isolates of strong gelatinase producer belong to genotype 1.The proportion of isolates with DNase,hemolysin,casein and gelatinase activities of T.asahii isolated from bloodstream infections,was higher than that of non-bloodstream infections.In this study,only five isolates were unable to form biofilms,including four T.asahii isolates and one T.dohaense isolates.The percentage of medium or strong biofilm producer is 73.2%,and the proportion weak biofilm forming producer in genotype 1(40%)is higher than that of genotype 3(12%,P=0.015)and genotype 4(11.8%,P=0.005).Regardless of the ability to form biofilm,the biofilm of Trichosporon isolates is highly resistant to all azoles,with MBIC50>1024?g/ml.In terms of the azole resistance mechanism,the efflux pump activity of clinical resistant isolate of 16G1229 was significantly increased.The xenobiotic-transporting ATPase encoding gene A1Q106164 and ATP-binding cassette(ABC)transporter Pdr11p encoding gene PDR11,the expression levels were induced by fluconazole with FPKM values of 367.94± 14.41 and 339.99± 16.9,respectively.We speculated this mechanism contributed to the increased efflux pump activity and azole resistance.The amino acid substitution S1343T in Pdrllp may contribute to the overexpression of PDR11.Regardless of the fluconazole pressure,the expression level of ERG 11 gene is significantly increased,which may play an important role in azole resistance.Conclusions:In the present study,the molecular epidemiology and virulence distribution invasive infections of Trichosporon were clarified in China.The distribution of antifungal susceptibility was investigated.Then we established the tentative ECV values accordingly.For the first time,the up-regulation expression level of efflux pump encoding genes and the overexpression level of EGR11,showed a potential role in the azole resistance of T.asahii.Background:Candida glabrata was the common pathogen causing invasive candidiasis.C.glabrata displayed an inherently reduced susceptibility to azoles,meanwhile,with a staggering rising in echinocandin resistance.Gcn5 is one of the most characterized fungal KATs,which is necessary for transcription regulation in eukaryotes.Here,we explore the role of GCN5 in antifungal resistance and pathogenicity in C.glabrata by genetic ablation of GCN5,as well as the expression dynamics of critical genes and pathways.Methods:The GCN5 gene in C.glabrata strain ATCC 2001 were knocked out and restored by using the Alt-R CRISPR-Cas9 system.Antifungal susceptibility testing and serial dilution growth assays were performed to detect the susceptibilities against antifungal drugs and stress agents of C.glabrata lacking GCN5.Time-kill kinetics of WT(ATCC 2001)strain and gcn5? strain against micafungin and fluconazole were carried out.Fungicidal and fungistatic activity were calculated,and resistant colony frequency and resistance-conferring mutations were evaluated.For western blot analysis,the effect of GCN5 deletion for the level of histone acetylation was detected.By transcriptomic analysis,we explored the regulatory mechanism of GCN5 deletion on drug resistance and virulence for C.glabrata.THP-1 macrophage infection assays were performed for detecting the viability of C.glabrata inside THP cells.Results:Deletion of GCN5 in WT background increases 2 to 4-fold susceptibility for echinocandins,2-fold for azoles and SCY048,more importantly,more than 8-fold for FK506(4 ?g/ml)+micafungin and APX001 A.Deletion of GCN5 appeared to complete or near complete reverse the Fks-mediates echinocandins resistance by FK506 in 4?g/ml.Disruption of GCN5 dramatically enhanced the fungicidal or fungistatic activity of micafungin and fluconazole.Reduced colony frequency and gain-of-function mutations detected from this dynamic experiment.Compared to the WT and gcn5?::GCN5 strain,the acetylation level of H3K9 and H3K14 in gcn5? strain were around 0.6-fold and 0.45-fold to those in WT strain.GCN5 deletion changes the landscape of several transcriptional networks by regulating specific functional gene group.The distribution of genes corresponding to GCN5 deletion major enriched in transmembrane transport,transmembrane transporter activity,component of membrane and plasma membrane and cell wall.Lack of GCN5 unable to drive the expression of transmembrane transporter activity genes,cell wall component synthesis genes and multidrug resistance transporters genes.Cells lacking GCN5 need to mobilize much more genes to sustain life upon micafungin pressure.Most of the significantly changed genes in gcn5? are down-regulated in response to micafungin treatment,which are major associated with cell wall biosynthesis.gcn5? cells were much more sensitive to killing by THP cells.Loss of GCN5 significantly reduced the viability of C.glabrata inside THP cells.Conclusions:C.glabrata histone acetyltransferase GCN5 plays a critical role in the antifungal resistance,stress response and pathogenesis.Loss of GCN5 attenuated antifungal drug resistance and pathogenicity in C.glabrata.We propose Gcn5 could be a promising candidate target for new drug discovery or therapeutic intervention to combat fungal infections. |
PDF Full Text Request