Study On The Role And Mechanism Of Minocycline In Cryptococcus Neoformans Infection

In recent years,the morbidity and mortality of invasive fungal infections have increased significantly.Cryptococcus neoformans is one of the most common pathogens of invasive fungal infection in humans.The common infection sites are the respiratory system and central nervous system,which can cause life-threatening meningoencephalitis.At present,there are some differences in the treatment of Cryptococcus neoformans recommended by domestic and foreign guidelines,but fluconazole is the most commonly used drug for the treatment and prevention of Cryptococcus neoformans infection.In recent years,the detection rate of fluconazole-resistant Cryptococcus neoformans has gradually increased because of its high frequency used in all the recommended schemes.Studies have shown that among the 4995 clinical isolates of 3210 patients,the fluconazole resistance rate of primary cases was 10.6%,and that of recurrent patients was 24.1%.Therefore,there is an urgent need to develop new alternative drugs for the treatment of Cryptococcus neoformans infection.It has been reported that MINO / FLU has a synergistic effect on clinical isolates of Candida albicans and Cryptococcus neoformans,but these studies are based on FLU-sensitive strains of Cryptococcus neoformans,so there is a lack of understanding of the effectiveness and mechanism of MINO / FLU combination against FLU-resistant Cryptococcus neoformans in vivo and in vitro.ObjectiveIn this study,H99 strain and FLU resistant strain(CN526)were used as the research objects.The drug sensitivity test in vitro,Galleria mellonella infection model,and CD-1 mice infection model were used to evaluate the effect of MINO alone or in combination with FLU on CN526 infection.The mechanism of the role of MINO in Cryptococcus neoformans was revealed by scanning electron microscope and transcriptome sequencing.These results will provide a preliminary theoretical basis for the combination of MINO / FLU in the treatment of FLU-resistant Cryptococcus neoformans.Methods(1)Clinical isolates of Cryptococcus neoformans were continuously passaged in FLU medium with different concentration gradients in vitro,and the strains of FLU-resistant Cryptococcus neoformans were screened in vitro.According to the literature method,the genetic stability of drug-resistant mutant colonies was tested,and the in vitro test showed that the genetic stability was reliable.(2)Antifungal activity of MINO and FLU against Cryptococcus neoformans was determined by CLSI M27-A3 micro broth dilution method,and the synergism between FLU and MINO was evaluated by chessboard micro broth dilution method.(3)Germicidal test was carried out in vitro,and the experiment was divided into four groups: control group,FLU group,MINO group,and MINO / FLU group.Each group was inoculated with H99(FLU sensitive)and CN 526(FLU resistant)strains.(4)Biofilm production ability of 15 strains and H99 strains were evaluated in vitro.The high-yield biofilm strains(CN18,CN117,CN526)and H99 strains were selected as the research objects,the ability of MINO / FLU to resist the pre-formed biofilm was tested,and the anti-biofilm effect of MINO / FLU was detected by CLSM.(5)The Galleria mellonella infection model of Cryptococcus CN 526 was established,and the survival and histopathological changes of CN 526 infected with MINO / FLU were evaluated.(6)CD-1 mice infection model of CN 526 was established.The survival of CN 526 infected mice treated with MINO / FLU was evaluated,and the fungal load of lung,liver,kidney,and brain of mice was evaluated,and the pathological changes of lung of mice was further analyzed.Finally,an enzyme-linked immunosorbent assay was used to detect the expression of inflammatory factors TNF-?,IL-6,IL-17 A,IL-10,and IL-22 in lung tissue of mice.(7)Ultrastructural changes of Cryptococcus neoformans(H99)treated with MINO were observed by scanning electron microscope.(8)Differentially expressed genes of Cryptococcus neoformans(H99)after MINO treatment were preliminarily discussed by the method of reference transcriptome sequencing analysis,and the results of transcriptome analysis were verified by qRT-PCR.Results(1)FLU-resistant Cryptococcus neoformans was screened in vitro and the drug-resistant mutant colonies' genetic stability was tested,and the results showed that the genetic stability was reliable,and the construction of FLU-resistant strains of Cryptococcus neoformans was successful.Among the 14 strains of Cryptococcus neoformans,5 strains of Cryptococcus neoformans(including H99)were sensitive to FLU,the MIC was between 1-4 ? / ml,and the other 9 strains were resistant to FLU.MIC of all 14 strains of Cryptococcus neoformans with MIC > 32 ?g / ml,to MINO >128 ?g / ml,showed that these strains were resistant to MINO in vitro.(2)In vitro chessboard micro-broth dilution method showed that the synergistic effect was observed in 9 strains of FLU-resistant Cryptococcus neoformans,and the FICI value was between 0.09 and 0.38.On the contrary,for the combined use of MINO/ FLU to FLU-sensitive strains,there was no difference in FICI value.When MINO /FLU was used in the treatment of FLU-resistant Cryptococcus neoformans,the use of MINO could greatly reduce the MIC value of FLU from > 32-128 ?g / ml to 8-32 ?g /ml,indicating that MINO can significantly improve the sensitivity of FLU-resistant Cryptococcus neoformans to FLU.Based on the FICI model,the FICI value of MINO /FLU combined therapy was 0.09-0.38,FICI < 0.5,indicating that MINO / FLU had a synergistic effect on FLU-resistant Cryptococcus neoformans.(3)Germicidal test in vitro showed that the effect of Cryptococcus neoformans(H99 and CN 526)treated with MINO or FLU was weak,and the effect lasted less than48 hours.Under the concentration of MIC of MINO or FLU,the effect on sensitive strains(H99)and drug-resistant strains(CN 526)was limited.In contrast,MINO / FLU combined treatment group showed a rapid and continuous effect on H99 and CN 526 during the experiment.Besides,in the MINO / FLU combined treatment group,the colony count of CN526(resistant strain)decreased more than 2 log10 CFU / ml,compared with that of H99(sensitive strain)at 72 h,and compared with the initial colony count of CN526,the colony count decreased by more than 3 log10 CFU / ml,and more than 3 log10 CFU / ml compared with the initial inoculation of CN526(0 h).The results showed that the combined treatment of MINO / FLU had a long-lasting effect on sensitive strains(H99)and drug-resistant strains(CN 526),and the effective effect on drug-resistant strains(CN 526)was more obvious.(4)Biofilm production ability of 15 strains and H99 strains were evaluated in vitro,and it was found that all strains were high biofilm-producing strains.Three high-yield biofilm strains(CN 18,CN 117,CN 526)and H99 strain were selected as the research objects.We found the biofilm with pre-formation time ? 36 h by CN 18,CN 117 and CN526,MINO could reduce the s MIC80 value of FLU from > 256 ug / ml to 8 × 64 ug / ml,FICI between 0.05 ug / ml and 0.38 ug / ml,FICI,and the FICI value were significantly less than 0.5,indicating that there was a strong synergistic effect between MINO and FLU.For the biofilm with pre-formation time > 36 h,there was no significant difference between MINO and FLU when s MIC80 was combined with FLU alone,and the FICI value was 2,indicating that the interaction between MINO and FLU was not obvious.The above data showed that MINO and FLU had a synergistic inhibitory effect on the formation of biofilm in the early stage(? 36 h),but had a poor synergistic inhibitory effect on mature biofilm(> 36 h).The results of CLSM detection also showed that MINO / FLU could synergistically inhibit the activity and growth of biofilm.(5)The Galleria mellonella model of CN 526 infected was successfully established,and the combined effect of MINO / FLU was evaluated by this model.The results showed that the 7-day survival rate of the MINO / FLU group was 35%,while that of the single drug group,whether MINO group or FLU group,was 0%.There was a significant difference in the 7-day survival rate between MINO / FLU group and MINO/ FLU group.After 4 days of infection,the survival rates of larvae treated with FLU,MINO,and MINO / FLU were 15%,0%,and 85%,respectively,indicating that MINO or FLU had a weak protective effect on larvae infected with CN 526.The results showed that the survival rates of larvae treated with MINO and FLU were 15%,0%,and 85%,respectively.The 7-day survival rate of the larvae infected by MINO / FLU was 35%,which indicated that the combined use of MINO / FLU could improve the7-day survival rate of the larvae infected by CN 526.(6)CD-1 mice infection model of CN 526 was established,the results showed that there was no improvement in the survival rate of CD-1 mice infected with CN 526 in the single drug treatment group,no matter in low MINO group,medium MINO group,high MINO group or FLU treatment group,but there was a significant difference in 27 days survival rate compared with medium MINO/FLU treatment group,indicating that MINO or FLU alone had a weak protective effect on CD-1 mice infected with CN 526.MINO/FLU combined therapy could protect CD-1 mice against CN 526 infection.The low MINO/FLU combined group could increase the survival rate of 27 days mice,but the survival rate was slightly lower than that of the medium MINO/FLU combined group.However,continuing to increase the dose of MINO cannot play a better protective effect.The results of the fungal load test showed that MINO/FLU combined treatment could significantly reduce the fungal load in tissues(lung,liver,kidney,brain)of CD-1 mice infected with CN 526.The pathological results also showed that MINO /FLU combined therapy had a protective effect on lung injury in CD-1 mice infected with FLU-resistant Cryptococcus neoformans.The detection of inflammatory factors in lung tissue showed that compared with the FLU monotherapy group,the expression level of TNF-? in the medium MINO group and medium MINO / FLU combination group decreased,and the difference was statistically significant(p < 0.01).The expression levels of IL-10 and IL-22 in the medium MINO group and medium MINO/FLU combined group were significantly higher than those in the FLU monotherapy group(p < 0.01).(7)Scanning electron microscope showed that there was a high density of yeast cells in the H99 group,forming lemon-shaped or oval cells,and a large number of budding yeast cells could be seen.However,in the H99+MINO group,the density of yeast cells decreased significantly,the cell body of yeast cells became larger and spherical,the walls became irregular and rough,showed wrinkled changes,and there were few budding yeast cells.(8)Sequencing results of reference transcriptome showed that the expression of235 genes in strain treated with H99+MINO was up-regulated and 126 genes down-regulated compared with H99.GO analysis showed that in the molecular biological function,it was mainly enriched in the secondary active transmembrane transporter activity,the hydrolase activity acting on the sugar bond,the hydrolase activity of hydrolyzing O-glycosyl compounds,and the active transmembrane transporter activity,while in the biological process,it was mainly enriched in the process of carbohydrate decomposition,carbohydrate metabolism and transmembrane transport.In the cytological components,it is mainly concentrated in the components of the membrane,the inherent components of the membrane,the membrane,and the plasma membrane.KEGG enrichment results showed that glycerol phospholipid metabolism,fructose and mannose metabolism,glycolysis/gluconeogenesis,DNA replication,base excision repair,MAPK signal pathway,ABC transporter,peroxisome,cell cycle,meiosis,endocytosis,and other pathways deserve our attention.Combined with the results of the scanning electron microscope,we focused on the genes related to the formation of the yeast cell membrane and transmembrane transport,such as ops1,nuc P,dcp2,ama T,mir T,mfs T,meb P and so on.Analysis of the above genes showed that the results of RNA-Seq and qRT-PCR of all the selected differentially expressed genes were consistent.Conclusions(1)MINO / FLU has a synergistic inhibitory effect on Cryptococcus neoformans resistant to FLU in vitro.(2)MINO / FLU has a synergistic inhibitory effect on the biofilm formed in the early stage but has a weak inhibitory effect on the biofilm in the late stage.(3)MINO / FLU has a protective effect on Galleria mellonella model infected by FLU-resistant Cryptococcus neoformans.(4)MINO / FLU has a protective effect on CD-1 mice infected by FLU-resistant Cryptococcus neoformans.(5)The mechanism of MINO in Cryptococcus neoformans may be related to the cell membrane of yeast.