| Backgrounds and Objectives:Trichosporon asahii(T.asahii)is an opportunistic pathogen which belongs to the members of basidiomycete yeast-like fungi and can cause fatal mycosis in immunocompromised patients with a high mortality rate(80%).Effective host immune response and antifungal susceptibility are two critical fators for the outcome of fungal infection.In this study,the early transcriptional response of human monocyte-like THP-1 cells to T.asahii infection was evaluated using c DNA microarray and may provide a foundation for further investigations into the pathogenesis of T.asahii infection.The phagocytosis by monocytes has been demonstrated to be significantly less efficient than macrophages.Monocytes only count for 3%-8% of peripheral blood leukocytes.Less numbers of monocytes may be not enough to resist the invasion and disseminated infection by T.asahii.Granulocyte-Macrophage Colony-Stimulating Factor(GM-CSF)plays an important role in regulating the proliferation,differentiation and activation of both neutrophiles and monocytes.We further investigated the effects of GM-CSF on the phagocytosis and fungicidal activities of human monocyte-like THP-1 cells against T.asahii infection.Invasive infections by T.asahii are usually associated with central venous catheters,vesical catheters,and peritoneal catheter-related devices.The ability of form biofilms on medical implanted devices may account for the resistance to antifungal drugs and results a high mortality rate.Invasive infections by T.asahii resistant to antifungal drugs have become increasingly common even with the combination of different types of antifungal drugs.However,combination of clinical commonly used antifungal drugs with non-antifungal agents may be a promising approach to cope with resistant T.asahii infections.In this study,the in vitro activities of combinations of sertraline or berberine hydrochloride with clinical commonly used antifungal drugs(fluconazole,voriconazole,itraconazole and amphotericin B)against planktonic forms and biofilms of clinical T.asahii isolates were tested by a broth microdilution checkerboard method and XTT reduction assay.Methods:Part I: Global gene expression profile response of human monocyte-like THP-1 cells to T.asahii infectionHuman monocytic THP-1 cells(1×107)were co-cultured with T.asahii(5×107CFU)or medium alone at the multiplicity of infection(MOI)of 5 for 3 h at 37℃ RPMI 1640 medium.After infection for 3 h,extracellular and non-adherent fungi were removed by washing twice with RPMI 1640.Total RNA was extracted from human monocytic THP-1 cells by using the Trizol reagent and microarray analysis was performed using a 35 k human whole-genome array(Capital Bio Corp,Beijing,China).To determine the significant differentially expressed genes,Significance Analysis of Microarrays(SAM,version 3.02)were used.Differentially expressed genes were analyzed by using DAVID Bioinformatics Resources version 6.7 to explore enriched Gene Ontology(GO)terms and enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway terms.Some selected differentially expressed genes were examined by q RT-PCR and ELISA to confirm the microarray data.Part II: Effects of GM-CSF on the activities of human monocyte-like THP-1 cells against T.asahii infectionHuman monocyte-like THP-1 cells(1×105/ml)were incubated with various concentrations of recombinant human GM-CSF(100,200,400U/ml)or medium alone for 48 h at 37℃,then co-cultured with T.asahii(5×105 CFU/ml)for 1 h.The phagocytosis activities of THP-1 cells against T.asahii were observed using an inverted microscope after stained with Wright-Giemsa stain.The fungicidal activities of THP-1 cells against T.asahii were assessed by count the number of colony forming units(CFU/ml).Part III: In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against planktonic forms and biofilms of clinical T.asahii isolates1.In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against planktonic forms of clinical T.asahii isolates:(1)21 clinical strains of T.asahii were used in the present study.T.asahii were resuspended in RPMI 1640 and adjusted to densities of 103 CFU/ml;(2)In vitro antifungal susceptibility tests were performed by a broth microdilution checkerboard method based on the CLSI M27-A3 broth microdilution method;(3)Determination of the minimum inhibitory concentration(MIC): The MIC values were recorded by using both MIC50(50% reduction in turbidity compared to the growth control well)and MIC100(complete inhibition of growth)endpoints;(4)Drug interaction analysis: The fractional inhibitory concentration index(FICI)was used to determine drug interactions.2.In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against biofilms of clinical T.asahii isolates:(1)21 clinical strains of T.asahii were used in the present study.T.asahii were resuspended in RPMI 1640 and adjusted to densities of 106 CFU/ml;(2)In vitro anti-biofilm susceptibility tests were performed using XTT reduction assay;(3)Determination of the sessile minimum inhibitory concentration(SMIC): The SMIC values were recorded by using both MIC50(50% reduction in turbidity compared to the growth control well)and MIC80(80% reduction in turbidity compared to the growth control well)endpoints;(4)Drug interaction analysis: The fractional inhibitory concentration index(FICI)was used to determine drug interactions.Results:Part I: Global gene expression profile response of human monocyte-like THP-1 cells to T.asahii infection1.The comparison of the global gene expression profile of T.asahii-infected and non-infected THP-1 cells revealed 1315 differentially expressed genes.Of these,463 genes were up-regulated,and 852 genes were down-regulated.A total of 479 GO terms and 21 KEGG pathway terms were enriched among up-regulated genes,while 514 GO terms and 18 KEGG pathway terms were enriched for down-regulated genes.Functional annotation clustering analysis revealed 30 GO annotation cluster and 1 KEGG pathway cluster among up-regulated genes,while 33 GO annotation cluster and 1 KEGG pathway cluster among down-regulated genes.The up-regulated genes were mostly involved in antifungal immune defenses,inflammatory responses,chemokine activity,cytokine activity,adaptive immune responses,as well as apoptosis and anti-apoptosis processes.The down-regulated genes during infection were predominantly related to apoptosis,cell cycle,mitosis,cell division and DNA repair.Genes encoding the pro-inflammatory cytokines and chemokines were strongly up-regulated.2.Seven seleted differentially expressed genes were examined by q RT-PCR and the results were consistent with microarray data.3.The ELISA results showed that THP-1 cells infected with T.asahii produced significantly more TNF-α,IL-1β and CCL3 proteins than did THP-1 cells cultured in medium alone and were consistent with microarray data.Part II: Effects of GM-CSF on the activities of human monocyte-like THP-1 cells against T.asahii infection1.The phagocytic rate of THP-1 cells to T.asahii(1 h)increased from 20% to 84% after pre-incubated with rh GM-CSF in a concentration-dependent manner ranging from 100 U/ ml to 400 U/ ml.2.The fungicidal activities of THP-1 cells were augmented after pre-incubated with rh GM-CSF in a concentration-dependent manner ranging from 200 U/ ml to 400 U/ ml.Part III: In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against planktonic forms and biofilms of clinical T.asahii isolates1.In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against planktonic forms of clinical T.asahii isolates:The MIC50 range of sertraline against 21 clinical T.asahii isolates was 4-8 μg/m L and the MIC100 range was 8-32 μg/ml.The MIC50 and MIC100 ranges of fluconazole,voriconazole,itraconazole or amphotericin B against T.asahii were obviously decreased when they combined with sertraline.The combinations of sertraline with fluconazole,voriconazole,itraconazole or amphotericin B showed significant synergistic effects(FICI≤0.5).The amphotericin B-sertraline combination showed the highest percentage of synergistic effects(90.5% of 21 clinical isolates).The MIC50 and MIC100 of berberine hydrochloride against 21 clinical T.asahii isolates were both higher than 128 μg/ml.However,the MIC50 and MIC100 ranges of fluconazole,voriconazole,itraconazole or amphotericin B against T.asahii were obviously decreased when they combined with berberine hydrochloride.The combinations of berberine hydrochloride with fluconazole,or amphotericin B showed significant synergistic effects(FICI ≤ 0.5).The amphotericin B-berberine hydrochloride combination showed the highest percentage of synergistic effects(71.4% of 21 clinical isolates).2.In vitro activities of combinations of sertraline or berberine hydrochloride with fluconazole,voriconazole,itraconazole and amphotericin B against biofilms of clinical T.asahii isolates:The SMIC50 range of sertraline against T.asahii biofilms was 16-32 μg/m L and the SMIC80 range was 32-64 μg/ml.The SMIC50 ranges of fluconazole or amphotericin B against T.asahii biofilms were obviously decreased when they combined with sertraline.The SMIC80 ranges of fluconazole,voriconazole,itraconazole or amphotericin B against T.asahii biofilms were not significantly changed.The amphotericin B-sertraline combination showed the highest percentage of synergistic effects against T.asahii biofilms(81% of 21 clinical isolates).The combinations of sertraline with fluconazole,voriconazole or itraconazole showed no significant synergistic effects against T.asahii biofilms(FICI>0.5).The SMIC50 and SMIC80 of berberine hydrochloride against T.asahii biofilms were both higher than 128 μg/ml.However,the SMIC50 and SMIC80 ranges of fluconazole or amphotericin B against T.asahii biofilms were obviously decreased when they combined with berberine hydrochloride.The SMIC80 ranges of fluconazole,voriconazole,itraconazole or amphotericin B against T.asahii biofilms were not significantly changed.The amphotericin B-berberine hydrochloride combination showed synergistic effects against T.asahii biofilms of 6 clinical isolates(28.6%).The synergistic effect of amphotericin B-berberine hydrochloride combination is uncertain because the synergistic percentage is very low and needs for further investigation.Conclusions:1.This study defines the early transcriptional response of monocyte-like THP-1 cells to T.asahii infection.The differentially expressed genes were mostly involved in antifungal immune defenses,as well as cell biologic processes.Genes encoding the pro-inflammatory cytokines(such as TNF-α and IL-1β),along with some chemokines(such as CCL3)were strongly up-regulated,suggesting that THP-1 cells can mount a powerful inflammatory response against T.asahii infection.2.This study indicates that rh GM-CSF can augment the phagocytosis and fungicidal activities of THP-1 cells against T.asahii.The application of rh GM-CSF may help to overcome the invasive T.asahii infections in immunocompromised patients.3.Sertraline exhibited excellent in vitro activities against both planktonic forms and biofilms of clinical T.asahii isolates in this study.The excellent anti-biofilms activitiy highlights the potential utility of sertraline on invasive T.asahii infections,especially suitable for the patients with indwelling medical devices.This study also suggests the potential utility of sertraline or berberine hydrochloride as synergist in combination with clinical commonly used antifungal drugs against T.asahii infection. |
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