Study On Potential Targets Of Antifungal Compounds By Monitoring Gene Expression Profiles

Dermatophytes belong to a group of closely related filamentous fungi that have the capacity to invade keratinized human and animal tissues to produce dermatophytoses. Global prevalence of dermatomycoses is as high as 20% according to a study by the WHO. Of these keratinophilic organisms, Trichophyton rubrum is the predominant causative agent for superficial dermatomycosis. The treatment of these cutaneous infections is based on the use of topical and systemic antifungal agents .Presently available antifungal agents are insufficient for the treatment of intractable T. rubrum infections and suffers from various drawbacks related to adverse effects and resistance,acting on limited targets and structural classes. Thus it is important to develop new antifungal agents that have potent activity against dermatophytes. The discovery of new drug targets is creating a bottleneck in antifungals development process.To screen potential targets of antifungal compounds, we developed a technology platform for high-throughput analysis and validation of mechanisms of action of lead compounds with potent activity against dermatophytes and of related potential target.In this experiment, the broth microdilution assay for antifungal and lead compound susceptibility testing of T. rubrum was previously developed as a modification of the National Committee for Clinical Laboratory Standards (NCCLS) M38-A method. Based on the M38-A method for broth dilution antifungal susceptibility testing of filamentous fungi, we determined effects of 6 kinds of solvents on coefficient of growth of Trichophyton rubrum and developed the M38-A method with some modifications, including inoculum density of conidia, medium, time and temperature of incubation, and endpoint assessment. The working concentration of every kind of solvent should be less than 1% (N,N-dimethylformamide), 8 % (polyglycol 200), 2 % (dimethyl sulfoxide), 4 %(acetone), 8%(dehydrated alcohol) and 2% (propylene glycol), respectively. An inoculum of 1×104-5×104 CFU/ml, a temperature of incubation of 28°C, an incubation time of 7 days, and 80% growth inhibition as the endpoint criterion, were the suitable testing conditions. The working concentration of every kind of solvent should be less than 1% (N,N-dimethylformamide), 8%(polyglycol 200), 2%(dimethyl sulfoxide), 4%(acetone), 8%(dehydrated alcohol) and 2% (propylene glycol), respectively. An inoculum of 1×104-5×104 CFU/ml, a temperature of incubation of 28°C, an incubation time of 7 days, and 80% growth inhibition as the endpoint criterion, were the suitable testing conditions. With reliable results and good reproducibility, the NCCLS 38-A method with some modifications is suitable for the method of broth microdilution assay for antifungal lead compound susceptibility testing of dermatophytes.At the same time, 14 cDNA libraries of T. rubrum were constructed and the cDNA plasmids were isolated and Sequencing was performed with a generic T7 primer located 5′upstream of the inserted segments. A total of 36,992 sequences were identified and 10,224 unique ESTs were isolated, including 4,566 contigs and 5,658 singletons. The clustered EST consensus sequences were assigned with potential functions through homologous comparisons by BLASTX searches of the GenBank non-redundant (NR) protein database. ESTs were further classified according to the NCBI Clusters of the Eukaryotic Orthologous Groups (KOGs). The metabolic pathways of T. rubrum were partially reconstructed by searching for known pathway homologs found in the Kyoto Encyclopedia of Genes and Genomes database (KEGG). A set of microarrays containing a total of 10,000 spots (8,997 clones in the form of PCR products and 1,003 controls, including blank, negative, and positive controls) were spotted in duplicate onto coated glass slides.Monitoring gene expression changes in response to a drug or chemical can help us to understand the mechanism of how drugs and drug candidates work in cells and organisms.Based on DNA microarray technology, gene expression responses of T. rubrum to two antifungals, itraconazole and clotrimazole, and a lead compound jatrorrhizine were studied by Monitoring gene expression profiles.The analysis of transcriptional profiles of T. rubrum in response to jatrorrhizine, a proberberine compound, revealed much information about mechanisms of action and molecular targets of jatrorrhizine. A total of 486 genes were found to be responsive to jatrorrhizine, including 234 up-regulated genes and 252 down-regulated genes. Of the genes that showed a response to jatrorrhizine, most were classified as"unknown function"(61.1%). Some others were classified as being involved in posttranslational modification, protein turnover, chaperones (4.73%), lipid, fatty acid, and sterol metabolism (4.1%), energy production and conversion (4.1%), amino acid transport and metabolism (3.7%), inorganic ion transport and metabolism (3.09%), Signal transduction mechanisms (2.88%), secondary metabolites biosynthesis, transport, catabolism(2.67%),RNA processing and modification(2.06%), and cell wall/membrane/envelope biogenesis(0.41%).Genes involved in lipid metabolism and especially in ergosterol biosynthesis were differentially expressed in response to jatrorrhizine, including upregulation of ERG6 and ERG5, and downregulation of ERG20, ERG11 and CBR1. The responsive genes ERG20 and ERG11 in this study are functional upstream of ERG11, suggesting that their repression is in response to overdose of intermediate products in their downstream. The responsive genes ERG5 in this study is functional downstream of ERG6, a putative target gene, and is functional upstream of terminal point of ergosterol pathway, suggesting that its induction is in response to ergosterol depletion. In addition, transcription of some genes involved in cell stress response, drug efflux, small molecule transport, and cell wall/membrane/envelope biogenesis was also affected by jatrorrhizine. Up-regulation of certain ion transport-related genes or down-regulation of others may be in response to destabilization of ionic homeostasis within the fungal cell when membranes are damaged by jatrorrhizine. Moreover, Ste11 coding for MAP kinase kinase kinase involved in osmotic stress was induced by jatrorrhizine, suggesting that the plasma membrane may be affected by this compound. Genes involved in chitin biosynthesis were also differentially expressed in response to jatrorrhizine, including upregulation of Chs7, Rho1, DW678390 coding for chitin synthase V and EL790146 coding for myosin type II heavy chain. The activity and localization of chitin synthase 3 (CHS3) is posttranslationally regulated. The responsive genes Chs7 was involved in the control of CHS3 export from the endoplasmic reticulum (ER). The responsive genes Rho1 was involved in regulating the transport of CHS3 from the trans-Golgi network/early endosome to the plasma membrane. At steady state, a proportion (50–70%) of Chs3p is maintained in a internal reservoir in the trans-Golgi network/early endosome.In this study, although we did not found gene coding for CHS3 was differentially expressed in response to jatrorrhizine,upregulation of Chs7 and Rho1 involved in regulating the activity and localization of CHS3 suggests CHS3 may be affected by this compound. Overexpression of gene coding for CHS3 does not lead to increased levels of CHS3 because the CHS3 excess is retained in the ER. Chitin synthase V plays an important role in chitin biosynthesis. Upregulation of DW678390 coding for chitin synthase V suggests chitin synthase V may be affected by this compound. Myosin heavy chain is involved in maintaining fungal cell budding and the deposition of chitin. Upregulation of EL790146 coding for myosin type II heavy chain suggests the quantity of chitin in cell wall may be decrease. Upregulation of genes involved in chitin biosynthesis suggests that cell wall was affected by jatrorrhizine. Some other proberberine compounds ininhibiting ERG6, CHS1 and CHS2 were reported in Candida albicans. All of these informations suggest that the mechanism of action of jatrorrhizine against T. rubrum may be involved in inhibiting ergosterol biosynthesis and chitin biosynthesis. Sterol 24-c-methyltransf- erase coded by ERG6, chitin synthase 3 and chitin synthase V may be potential targets of this compound.To validate microarray data, real time quantitative RT-PCR experiments were performed for 9 target genes ,including ERG6,with the same cDNA preparations used in array hybridizations. In general, the RT-PCR data showed good agreement with the microarray results, and there was a strong positive correlation (r = 0.99) between the two methods.In total,670 genes were differentially expressed upon exposure to itraconazole; 306 of which showed a significant increase in expression and 367 of which showed a significant decrease in expression. Of the genes that showed a response to itraconazole, most were classified as"unknown function"(57%). Others were classified as being involved in amino acid transport and metabolism (7.13%), posttranslational modification, protein turnover, chaperones (4.16%), translation, ribosomal structure and biogenesis (3.86%), lipid, fatty acid, and sterol metabolism (3.57%), energy production and conversion (3.42%), inorganic ion transport and metabolism (2.53%), signal transduction mechanisms (2.53%), and secondary metabolites biosynthesis, transport, catabolism(2.38%). Genes involved in lipid metabolism and especially in ergosterol biosynthesis were up-regulated in response to itraconazole, including ERG6, ERG7, ERG11, ERG24, ERG25 and ERG26. The gene encoding the target of the azoles, ERG11 was found to be up-regulated 13.55-fold. With the exception of ERG7, most of the responsive genes in this study are functional downstream of ERG11. This regulatory manner of gene expression in the ergosterol pathway suggests that their induction is in response to ergosterol depletion. The gene expression pattern changes produced by itraconazole were consistent with the known inentical mechanisms of action. The response of T. rubrum to clotrimazole and itraconazole was similar. To validate microarray data, real time quantitative RT-PCR experiments were performed.To sum up, we developed a technology platform for high-throughput analysis and validation of mechanisms of action of lead compound with potent activity against dermatophytes.The mechanism of action of jatrorrhizine against T. rubrum may be involved in inhibiting fungal ergosterol biosynthesis and chitin biosynthesis by analysis of gene expression profiles in response to jatrorrhizine. Sterol 24-c-methyltransferase coded by ERG6, chitin synthase CHS3 and chitin synthase V may be potential targets of the compound. Based on this technology platform, we validated the mechanism of action of itraconazole and clotrimazole by analysis of gene expression profiles in responses of T. rubrum to these two antifungals.