Quantitative Real Time PCR study on the effects of hilA gene deletion on the expression of pathogenicity genes in Salmonella enterica ssp.

Abstract

Salmonella enterica serovar Typhimurium is one of the important worldwide health issues. The hilA gene encodes a transcriptional activator that regulates expression of the majority of the genes responsible for the salmonella invasive phenotype. In the present study, PCR was performed using our designed primers for amplification of upstream and downstream regions of Salmonella Typhimurium hilA gene. Each DNA fragment was T/A-cloned into pGEM-T easy vector and then sub-cloned into pET32 expression vector together with Kanamycin resistance gene. Recombinant plasmid was transformed to bacteria (Salmonella Typhimurium) using electroporation. The hilA-Knockout mutant was characterized to evaluate the predicted role of the hilA gene in virulence. Quantitative RT-PCR was carried out to study the impact of hilA knock out on the expression of downstream genes including invF and invA. We confirmed the successful preparation of hilA gene construct (pET32-up-kan-down) followed by the efficient electroporation of the construct to bacterial cells. The homologous recombination resulted in the hilA and confirmed by PCR. We demonstrated that hilA knock out leads to attenuation of invA and invF genes expression. The hilA knockout strain may be useful for development of efficient vaccines against the Salmonella Typhimurium.