Metalloprotease Genes Expression in Trichophyton mentagraphytes and Trichophyton simiiContains Genetic Variations Isolated from Iraqi Patients Resistance to Ketoconazole and Amphotericin B

Abstract

This study aimed to use Real-Time Polymerase Chain Reaction analysis, to discover the metalloprotease genes (MEP1-5) in Trichophyton menagraphytes and Trichophyton simii contained Genetic variations and determine their expression and association with the antifungal resistance of dermatophytes. Two species of dermatophytes (five isolates) were used in this study (diagnosed morphologically and molecularly by ITS region previously), which included the following types are (Trichophyton mentagrophytes, and Trichophyton simii) of them contained variations of the percentage of conformity with the gene bank 99% and they were registered with the gene bank. The two species were tested for antifungal susceptibility by using the disk method. RNA was isolated from sample according to the protocol of TRIzol™ Reagent The genes used in this study were the metalloprotease gene (MEP1-5). The Livak method was used for determining gene expression. All the genes were amplified and normalized to 18S-Rrna as reference genes. All Trichophyton simii isolates contains the five genes MEP 1 to 5 but Trichophyton mentagrophytes one isolate contains five genes (MEP1-5) and others isolate contains four gene MEP 1 to 4 . It has been found that Amphotericin B inhibits the MEP 4 and MEP 5 genes other than the MEP1,2,3 genes. The MEP2 and3 recorded the highest increase in gene expression after treatmentin in Trichophyton simii , while in Trichophyton mentagrophytes the MEP1 recorded the highest increase in gene expression after treatment fllowed by MEP2,3,4 respectively. It has been found that Amphotericin B, had a better effect to inhibit the genes of fungi of Trichophyton mentagrophytes, and Trichophyton simii other than Ketoconazole, especially the MEP 5 followed by MEP 4.