Cloning and characterization of genes differentially expressed in pythium insidiosum Thai strain

Abstract

An emerging aquatic fungal like organism, Pythium insidiosum, causes the disease called ‘pythiosis’ in both human and animal from tropical, subtropical, and temperate zone. The highest mortality and morbidity in human pythiosis has been reported from Thailand. Very few genetic information and pathogenesis concerning this organism is not known. This study was to investigate genes expressed from culture at 37°C might involved in its pathogenesis. To isolate temperature–induced genes, PCR – select cDNA subtraction from cultures P. insidiosum at 27°C and 37°C and PCR-based searching the genetic information in GenBank Databases. Both subtracted cDNA libraries conditions, total six hundred and six clones with inserted cDNA fragments were detected. Nucleotide analysis by BLASTN showed mostly mitochondria DNA whereas the protein function predicted by Swiss-Prot and BLASTX programs demonstrated candidate genes encoding proteins. Here, gene encoding cytochrome c oxidase subunit II (COX II), β-tubulin (TUB), and chitin synthase subunit II (CHI II) were selected to analyze their expression using semi–quantitative PCR and confirmed by Real time RT–PCR. To demonstrate the expression level at 37°C and 27°C conditions, sixteen strains of P. insidiosum, represented three phylogeographic preference were recruited. The results showed that at 37°C growth condition, only COX II gene expressed 2.5 times over 27°C growth condition (p = 0.0347). Even though, TUB and CHI II show indistinguishable expression, these genes can be used as housekeeping genes for temperature susceptible gene expression studies. This is the new findings. However, it is of noted that the expression of CHI II at 37°C depended on the geographic distribution. Not only the expression was analyzed, the phylogenetic relationship was also performed using COX II. The result showed that COX II is the good candidate gene to reveal the genetic association among P. insidiosum. It also is an alternative gene of ITS which is commonly used for study the phylogenetic distribution. In conclusion, these data was the pioneer to demonstrate that COX II expressed at 37°C over 27°C. The insight of this gene and pathogenesis required to perform the further investigation.