Objective:To evaluate the effect of microevolution on phenotypes and drug resistance of the Trichosporon asahii biofilm. Methods:The standard strain of Trichosporon asahii was obtained from the Fungal Biodiversity Institute of the Royal Netherlands Academy of Arts and Sciences, the fluconazole-sensitive primary strain (TO) of Trichosporon asahii was isolated from a case of trichosporonosis diagnosed in the Department of Dermatology, the Seventh Medical Center of Chinese People′s Liberation Army General Hospital in 2000, and the fluconazole-resistant evolved strain (TEVO) of Trichosporon asahii was isolated from the above patient in 2014. Biofilms of the above-mentioned strains were formed in vitro, and tetrazolium salt XTT reduction assay was performed to evaluate growth kinetics of the Trichosporon asahii biofilm, and laser scanning confocal microscopy to determine the thickness of the biofilm; the sessile minimum inhibitory concentrations (SMICs) of fluconazole, itraconazole and voriconazole against the biofilms at different growth stages were determined in vitro for the evaluation of the resistance of the biofilms. One-way analysis of variance was used for comparisons among multiple groups, and Hartley test for testing homogeneity of variance. If the variance was homogeneous, least significant difference test was used for multiple comparisons; if the variance was heterogeneous, Tamhane′ T2 test was used for multiple comparisons. Results:In the adhesion (0 h) and formation stages (4- 24 hours) of the Trichosporon asahii biofilm, the metabolic activity of the evolved strain TEVO was the weakest (adhesion stage: F = 35.705, P < 0.001; formation stage: F = 15.042, P < 0.001) . At 48 hours after adhesion, the biofilms matured, and the TO strain showed the weakest metabolic activity ( F = 10.985, P < 0.001) . In the maturation stage, the biofilm thickness of the TEVO strain (26.1 ± 1.18 μm) was significantly higher than that of the TO strain (22.8 ± 1.73 μm, P = 0.001) , but significantly lower than that of the standard strain (29.5 ± 1.28 μm, P = 0.001) . As drug susceptibility testing showed, the SMICs of azole antifungal agents against the TEVO strain were higher than those against the TO strain in the adhesion and formation stages of the Trichosporon asahii biofilm, and the SMICs of azole antifungal agents against the biofilms of the 3 strains of Trichosporon asahii were all over 1 024 mg/L in the maturation stage of the biofilm. Conclusion:Under the dual pressure of host environment and antifungal drugs, adaptive changes took place in the phenotypes of the Trichosporon asahii biofilm with an increase in the resistance to azole antifungal drugs.

The opportunistic fungal pathogen Trichosporon asahii (T. asahii) is an important causal agent of mortality in immunocompromised patients and associated with frequent relapses, even with sufficient antifungal treatment. Investigating the proteomes of initial and recurrent isolates may help to identify within-host adaptive changes. In this study, using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) technology, we analyzed the proteomes of two T. asahii strains that were isolated 15 years apart from the same patient who suffered initial and recurrent episodes of systemic disseminated trichosporonosis. A total of 597 differentially expressed proteins were identified. Functional analysis showed that the increased proteins were primarily concentrated on peptide/protein/energy/drug metabolism and translation. Most of the results were determined to be consistent with the findings of phenotypic assays, such as tests for drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays. Moreover, we performed multiple reaction monitoring (MRM) mass spectrometry to verify 27 candidate proteins, and the results of this experiment were also highly consistent with the results of the TMT analysis. Therefore, to the best of our knowledge, these data provide the first molecular evidence of how the T. asahii proteome changes related to host-specific adaptation during human infection. Significance: Systemic infection with Trichosporon asahii (T. asahii) has recently been recognized as an important causal agent of mortality in immunocompromised patients. Although triazole treatment usually works efficiently in the early phase of infection, many patients relapse. Hence, comparative analyses of the proteomics of initial and recurrent isolates may reveal evidence of adaptive changes within the host. Our study demonstrates that the recurrent strain has undergone proteomic changes using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Moreover, the results of phenotypic assays, including drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays, were highly consistent with the proteomic changes, and multiple reaction monitoring (MRM) verification also showed similar trends to the TMT results. In summary, our study is the first to investigate the adaptation of T. asahii under pressure from antifungal chemotherapy and host immune responses.

Trichosporon is a yeast-like basidiomycete, a conditional pathogenic fungus that is rare in the clinic but often causes fatal infections in immunocompromised individuals. Trichosporon asahii is the most common pathogenic fungus in this genus and the occurrence of infections has dramatically increased in recent years. Here, we report a systematic literature review detailing 140 cases of T. asahii infection reported during the past 23 years. Statistical analysis shows that T. asahii infections were most frequently reported within immunodeficient or immunocompromised patients commonly with blood diseases. Antibiotic use, invasive medical equipment and chemotherapy were the leading risk factors for acquiring infection. In vitro susceptibility, clinical information and prognosis analysis showed that voriconazole is the primary drug of choice in the treatment of T. asahii infection. Combination treatment with voriconazole and amphotericin B did not show superiority over either drug alone. Finally, we found that the types of infections prevalent in China are significantly different from those in other countries. These results provide detailed information and relevant clinical treatment strategies for the diagnosis and treatment of T. asahii infection.

Objective To investigate differences in the expression of Ras 1,Rac1 and Rho1 genes between yeast and hyphal phases of Trichosporon asahii (T.asahii),and to explore their roles in the formation of hyphae.Methods The yeast phase and hyphal phase of T.asahii were cultured and served as yeast phase group and hyphal phase group respectively.Total RNA was extracted from the 2 groups,and real -time fluorescence-based quantitative PCR (RT-PCR) was performed to measure the mRNA expression of Ras1,Rac1 and Rho1.Results The hyphal formation rate was significantly lower in the yeast phase group than in the hyphal phase group (0.40％ ± 0.53％ vs.99.33％ ± 0.57％,t =13.93,P ＜ 0.05).When the mRNA expression of Ras1,Rac1 and Rho1 in the yeast phase group was all set as 1,that in the hyphal phase group was 25.17 ± 10.99,16.81 ± 7.80,42.61 ± 18.50,respectively,with significant differences between the two groups in the three parameters (t =3.81,3.51,3.90,respectively,all P ＜ 0.05).Conclusion Ras1,Rac1 and Rho1 genes may participate in the regulation of hyphal formation in T.asahii.

Trichosporon asahii (T. asahii) has emerged as a dangerous pathogen that causes rare but life-threatening infections. Its resistance to certain antifungal agents makes it difficult to treat, especially for patients undergoing long-term antibiotic therapy. In this study, we performed a series of fluconazole (FLC) perturbation experiments for two T. asahii strains, a clinical isolate stain CBS 2479 (T2) and an environmental isolate strain CBS 8904 (T8), to uncover potential genes and pathways involved in FLC resistance. We achieved 10 transcriptomes of T2 and T8 that were based on dose and time series of FLC perturbations. Systematic comparisons of the transcriptomes revealed 32 T2 genes and 25 T8 genes that are highly sensitive to different FLC perturbations. In both T2 and T8 strains with the phenotype of FLC resistance, the processes of oxidation-reduction and transmembrane transport were detected to be significantly changed. The antifungal susceptibility testing of FLC and penicillin revealed their resistance pathways are merged. Accumulated mutations were found in 564 T2 and 225 T8 genes, including four highly mutated genes that are functionally related to the target of rapamycin complex (TOR). Our study provides abundant data towards genome-wide understanding of the molecular basis of FLC resistance in T. asahii.

Superoxide dismutase (SOD) and catalase are considered the most important antioxidant enzymes which protect fungus from the oxidant damage of reactive oxygen species. In this study, we collected 44 strains of Trichosporon asahii (T. asahii) from different sources and investigated their SOD and catalase activities. The results showed that the SOD and catalase activities of Clinical group were significantly higher than those of Environment group (p < 0.01). The SOD and catalase activities of T. asahii in Internal passage group went up gradually after passage in mice, and were significantly higher in 5th generation of Internal passage group (p < 0.05). The SOD and catalase activities of Fluconazole-resistant group strains also increased after resistant induction, and the SOD and catalase activities were significantly higher in the 10th generation of Fluconazole-resistant group (p < 0.05). This implied that T. asahii has stronger antioxidant ability. The strains of T. asahii from different sources have different antioxidant abilities, which mainly manifest in the difference of antioxidant enzymatic activities. Clinical group strains have the strongest antioxidant capacity; Internal passage group strains and Fluconazole resistant group strains better; Environmental group strains the lowest. These results also suggested that the antioxidant defensive response of T. asahii might be relevant to its infection mechanism and drug resistance mechanism.

Objective To investigate the role of gene expression of Cdc42( cell division cycle42) in hyphal formation by detecting the differences of Cdc42 mRNA expression between yeast and hyphal forms of Trichosporon asahiis. Methods Three groups under different conditions were set up: one group including two subgroups of yeast and hyphal forms of type strain,one group including four subgroups of different growth times of type strain and one group including four subgroups of four strains of T. asahii isolated from different sources. Extracted the total RNA separately and measured the mRNA expression of Cdc42 with fluorescent quantitative PCR assay,then calculated the relative expression of mRNA. Results Different conditions resulted in different hyphal formation rates and Cdc42 expressed in all groups. With the increase of the rate,the expression of Cdc42 was almost increasing. Conclusion Cdc42 participates in the regulation of hyphal formation in T. asahii.

The aim of the current study was to observe the effects of suppressor of cytokine signaling 1 (SOCS1) silencing in human melanoma cells on cell biological behavior and interferon- (IFN-) sensitivity, and to investigate the use of SOCS1 as a therapeutic target in the treatment of melanoma. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to verify that SOCS1 interference effectively silenced the expression of SOCS1 in the Mel526 human melanoma cell line. For IFN- stimulation, western blot analysis was used to observe changes in expression levels of signal transduction and transcription activator (STAT) 1 and phosphorylated STAT (pSTAT) 1. Changes in the expression levels of IFN- regulatory factor 1 (IRF-1) were measured with RT-qPCR. Changes in the sensitivity of melanoma cells to IFN- were detected using an MTT assay. The cell proliferation rate was observed by cell counting and changes in the cell cycle were detected with flow cytometry. The results revealed that SOCS1 interference effectively silences SOCS1 expression in Mel526 cells. However, the S stage of the cell cycle was markedly extended. Following the inhibition of SOCS1 expression, the proliferation experiment demonstrated that the proliferation ability of Mel526 cells was decreased. Following IFN- stimulation, the expression levels of pSTAT and IRF-1 increased significantly compared with those in the controls. The MTT experiment showed that SOCS1 interference caused the median inhibitory concentration (IC50) of oxaliplatin in Mel526 cells to decrease significantly. In conclusion, SOCS1 interference reduced the proliferation ability of Mel526 human melanoma cells and increased their sensitivity to IFN-.

Angiotensin II (AngII) is a hormone with long-established cardiovascular actions. Previous studies have revealed an additional role for AngII in the regulation of cutaneous wound healing. To evaluate the association between AngII and abnormal pigmentation of cutaneous wound healing, the present study used human melanocytes to investigate the effects of AngII on melanogenesis, and to elucidate the possible underlying mechanisms. Primary culture melanocytes were treated with AngII either alone or in combination with an AngII type 1 (AT1) receptor antagonist, losartan (LOS). The melanin content and tyrosinase activity were measured and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to assess the proteins involved in melanogenesis and the AT1 receptor. AngII regulated the mRNA expression of AT1 in the melanocytes. The melanin content and tyrosinase activity increased in response to treatment with AngII in a concentration‑dependent manner. RT-qPCR and western blotting revealed that the AT1 receptor antagonist, LOS, eliminated this effect. These results provide a novel insight into the role of AngII and its associated signaling in melanogenesis.

Trichosporon asahii is the major cause of invasive trichosporonosis, but little is known about the host immune response to this pathogen. In this study, the early transcriptional response of human monocyte-like THP-1 cells to T. asahii infection was evaluated using cDNA microarray and 1,315 differentially expressed genes were identified. The up-regulated genes were mostly involved in both innate and adaptive immune responses, as well as apoptosis and anti-apoptosis processes. Genes encoding the pro-inflammatory cytokines TNF-α, IL-1β, IL18 and IL-23α, along with the both C–C motif and C–X–C motif chemokines were strongly up-regulated, suggesting that THP-1 cells can mount a powerful inflammatory response to T. asahii infection. Genes encoding pattern recognition receptors were found up-regulated, such as dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin, cluster of differentiation 36 and the long pentraxin 3. Genes encoding members of the dual-spasticity phosphates family were up-regulated, and these genes were considered as a negative feedback mechanism to prevent excessive inflammatory response. The down-regulated genes in T. asahii -infected THP-1 cells were predominantly associated with cell cycle, mitosis, cell division and DNA repair. Thus, our study defines the early transcriptional response of monocyte-like THP-1 cells to T. asahii infection and provides a foundation for further investigations into the pathogenesis of T. asahii infection.

目的研究RAW264.7小鼠巨噬细胞与T.asahii相互作用后TNF-α,IL-1β和CCL3的表达水平变化。方法RAW264.7细胞与T.asahii菌液共孵育3 h、6 h、9 h、12 h,对照组不加菌液。在各孵育时间点取上清液,采用双抗体夹心酶联免疫吸附试验（ELISA）试剂盒检测上清液中TNF-α、IL-1β和CCL3的含量。台盼蓝拒染法计各孵育时间点RAW264.7细胞的活细胞数。结果 RAW264.7细胞与T.asahii共孵育3 h、6 h、9 h、12 h后,TNF-α、IL-1β、CCL3的表达水平均明显高于对照组,有显著性差异（P＜0.05）。3种细胞因子的表达水平随共孵育时间延长而增高,共孵育9 h的表达水平最高。RAW264.7细胞的活细胞数随共孵育时间延长而逐渐下降,共孵育12 h后的活细胞数量与其他各时间点有统计学差异（P＜0.05）。结论 RAW264.7小鼠巨噬细胞面临T.asahii感染时可分泌TNF-α、IL-1β、CCL3等细胞因子,这些炎性细胞因子可能参与了抗T.asahii感染的免疫反应。

Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future.

目的 了解阿萨希毛孢子菌(Trichosporon asahii,T.asahii)菌丝生长与顶体(spitzenk(o)rper)的关系.方法 用荧光染剂FM4-64对YPD液体培养基培养后的T.asahii进行荧光染色,经不同浓度细胞松弛素D抑制后,在荧光显微镜下观察染色结果.结果 在T.asahii芽胞、芽管以及菌丝顶端和亚顶端均可见到FM4-64荧光聚集；随着细胞松弛素D浓度的增加,其顶体荧光逐渐消失.T.asahii的生长受到明显抑制.结论 T.asahii存在与真菌极性生长密切相关的顶体,其在控制菌丝生长方面起着重要的作用.

Objective To induce fluconazole resistance in T.asahii by culture in medium containing increasing concentrations of fluconazole,and to evaluate the stability of the induced resistance.Methods Two T.asahii strains with a highest sensitivity to fluoconazole,including a clinical isolate CBS2479 (minumum inhibitory concentration (MIC) =0.25 μg/ml) and an environmental isolate CBS8904 (MIC =1.5 μg/ml),were selected from 11 T.asahii strains stored in the laboratory of the Department of Dermatology,General Hospital of Beijing Military Region.Both strains were respectively and serially subcultured in potato dextrose agar (PDA) medium containing growing concentrations of fluconazole (from 0.5 MIC to 256 μg/ml).E-test was performed to evaluate the susceptibility of T.asahii to fluconazole after each passage.To evaluate the stability of fluconazole resistance,the T.asahii isolates with induced resistance (MIC ＞ 256 μg/ml) were serially subcultured in drug-free PDA medium,and drug susceptibility assay was performed after each subculture.Results After serial culture in PDA medium containing fluconazole,high level of fluconazole resistance (MIC ＞ 256 μg/ml) developed in both of the fluconazole-susceptible T.asahii strains CBS2479 and CBS8904.The MIC value of fluconazole remained unchanged in the fluconazole-resistant strain CBS2479R,but gradually decreased to 64 μg/ml in the other resistant strain CBS8904R after 18-day culture in fluconazole-free PDA medium.Conclusions Fluconazole resistance can be induced in T.asahii strains from different origins by serial culture in medium containing growing concentrations of fluconazole,and the stability of the induced fluconazole resistance varies between strains of different origins.

Trichosporon asahii is one of the important opportunistic pathogenic fungi. Here, we first report the draft nuclear chromosome genome sequence and mitochondrial genome sequence of T. asahii CBS 2479, which is a standard strain of T. asahii that was isolated from a progressive psoriatic lesion. COG analysis predicted that 3,131 genes were assigned to 23 functional categories and that 628 genes were predicted to have a general function.

ABSTRACT This is the first report of the genome sequence of Trichosporon asahii environmental strain CBS 8904, which was isolated from maize cobs. Comparison of the genome sequence with that of clinical strain CBS 2479 revealed that they have >99% chromosomal and mitochondrial sequence identity, yet CBS 8904 has 368 specific genes. Analysis of clusters of orthologous groups predicted that 3,307 genes belong to 23 functional categories and 703 genes were predicted to have a general function.

Objective To determine the effect of interferon-gamma (IFN-γ) on the phagocytosis and killing activity of a murine macrophage cell line RAW264.7 against T.asahii,and to estimate the possibility of treating T.asahii infection with IFN-γγ.Methods T.asahii was cultured with or without the presence of different concentrations (10,100,1000 U/ml) of IFN-γfor 18 hours followed by the incubation with RAW264.7 cells for different durations.After additional culture for 45 minutes,the number of T.asahii cells phagocytosed by RAW264.7 cells was counted under an inverted microscope,and the rate of phagocytosis was calculated.The number of colony forming units of T.asahii per milliliter (cfu/ml) was counted after 4-hour additional culture and the growth inhibition rate was determined.Data were processed by the SPSS 16.0 software,and comparisons of parameters between these groups were done by Bonferroni method and analysis of variance after homogeneity test of variance.Results The number of phagocytosed T.asahiicells was 25.12 ± 1.81,35.88 ± 3.56,52.12 ± 3.23,with the phagocytosis rate being 25.12％,35.88％ and 52.12％,respectively in RAW264.7 cells incubated with IFN-γ of 10,100,1000 U/ml,significantly different from that in untreated RAW264.7 cells (13.62 ± 2.39,13.62％,all P ＜ 0.01).The colony forming units of T.asahii per ml after incubation with untreated RAW264.7 cells differed significantly from those after incubation with IFN-γ (10,100,1000 U/ml)-treated RAW264.7 cells ((68.12 ± 3.39) × 500 vs.(58.62 ± 4.89) × 500,(45.50 ± 3.02) × 500 and (34.62 ± 4.24) × 500,all P＜0.01),with the growth inhibition rate being 25.21％,41.95％ and 55.83％ respectively for RAW264.7 cells incubated with IFN-γ of 10,100,1000 U/ml.Statistical differences were also observed in the killing activity between RAW264.7 cells incubated with different concentrations of IFN-γ (all P ＜ 0.01).Conclusion IFN-γ (10-1000 U/ml) may enhance the phagocytosis and killing activity of RAW264.7 cells against T.asahii in a concentration-dependent manner.

目的观察不同浓度壳聚糖对阿萨希毛孢子菌（asahii trichosporon,T.asahii）生物被膜（biofilm,BF）形成的影响。方法以T.asahii聚苯乙烯表面形成生物被膜48h为评价标准,观察其在0.25mg/ml,0.50mg/ml,0.75mg/ml,1.0mg/ml,1.25mg/ml和1.5mg/ml壳聚糖溶液下观察T.asahii BF的差异性;并应用XTT法测定生物膜活性并定量分析。结果 48h内0.25mg/ml,0.5mg/ml,0.75mg/ml和1.0mg/ml壳聚糖溶液没有明显抑制生物被膜形成的作用;1.25mg/ml壳聚糖溶液明显具有抑制T.asahii BF形成的作用,96h后逐渐消失抑制作用。结论一定浓度壳聚糖可明显抑制生物被膜的形成,但具有一定时效性。

MicroRNAs are short, 18- to 25-nt sequences of noncoding, single-stranded RNA that function as regulatory molecules and participate in a series of vital processes including early development, cell proliferation, cell differentiation, apoptosis, substance metabolism and the pathogenesis of human diseases. This study compared the microRNA profiles of patients with systemic scleroderma (SSc) and healthy control individuals to investigate the pathogenesis of SSc. Skin tissue was isolated from three patients with SSc and three healthy controls. miRNA microarray chip analysis identified 24 miRNAs that were differentially expressed in patients with SSc and 6 microRNAs that may be correlated with the pathogenesis of SSc. The results of the microarray analysis were confirmed using real-time PCR. This work suggests that miRNAs may be potential diagnosis biomarkers and are likely to be involved in the pathogenesis of SSc.

To investigate the hyphal-form transition of Trichosporon asahii (T. asahii) of different sources under different temperatures, media and cultural times; eleven T. asahii strains were isolated and maintained in RPMI-1640 medium, YPD liquid medium, Tween yeast bouillon (TYB) medium and 50% (v/v) fetal calf serum (FBS) medium independently to induce the hyphal growth. Culture was performed at 15, 25 and 37 degrees C for 1, 2, 3, 6, 12 and 24 h and the hyphal formation (growth) rate was calculated. The mean hyphal formation rate was (0.15 +/- 0.42)% at 15, (5.75 +/- 3.48)% at 25 degrees C and (33.81 +/- 15.39)% at 37 degrees C (P<0.05 for all) at the same time point. The mean hyphal formation rate was (46.24 +/- 25.50)% in RPMI-1640, (36.28 +/- 21.85)% in YPD, (33.93 +/- 21.29)% in TYB and (18.60 +/- 14.58)% in 50%(v/v) FBS showing significant difference intergroups (F = 6.29, P = 0.0013) and intragroups (F = 21.80, P = 0.0000). Moreover, the mean hypha formation rate also raised in all T. asahii strains with the prolongation of induction. Among various conditions for hypha induction of T. asahii, culture in RBMI1640 liquid media at 37 degrees C for 24 h is the best condition in which the hypha with high purity can be collected. The source of fungus is also an important factor influencing the hyphal growth of T. asahii.