Avaliação da regulação transcricional do fator sigma E de Corynebacterium pseudotuberculosis em resposta aos estresses nitrosativo e ácido

Abstract

Corynebacterium pseudotuberculosis is an intracellular pathogenic bacteria of great veterinary importance that infects small ruminants, causing a disease known as Caseous Lymphadenitis. This disease is distributed worldwide and has been associated with economic losses. Although the pathogenesis of this disease is well understood, there are few studies on the molecular determinants of pathogenicity of this microorganism. In this context, it Ls required new studies on the biological mechanisms involved during the course of infection, mainly because there are no satisfactory treatment and vaccines to fight the disease yet. In order to understand the role of the determinants of pathogenicity and how they are regulated during the process of infection, our research group has focused in proteins that modulate gene expression such as sigma factors of bacterial RNA polymerase. The regulation of gene expression by alternative sigma factors is crucial for adaptation and survival of intracellular pathogens by promoting the transient activation of specific genes involved in response to hostile conditions encountered in intrafagossomic environment, generated by the host immune system. In C. pseudotuberculosis, ÐE is the most well studied alternative sigma factor and it has been demonstrated its role in the adaptive response of this microorganism. However, it has not been carried out a detailed study on the regulation of expression of the sigE gene, which encodes sigma factor E. This work was done a characterization of the structure of the transcriptional unit of this gene using bioinformatics algorithms and by RT-PCR method in an attempt to confirm whether the gene sigE of C. pseudotuberculosis 1002 would be arranged in an operon in conjunction with the genes cseE and tatB, which encode, respectively, a possible negative regulator of sigma factor E and a component of an alternative secretion system, the Twin Arginine Translocase System. Moreover, the level of transcription of the genes sigE, tatB and cseE were evaluated by RT-qPCR essays in cultures exposed to nitrosative and acid stresses in order to confirm their involvement in adaptive response to environmental stress. The results of this study confirmed that the genes sigE, cseE and tatB constitute an operon in this species and that the genes sigE and cseE are activated in response to nitrosative and acidic stress while tatB is only activated in response to nitrosative stress. Taken together, these data suggests that the operon genes could be differentially regulated, by a complex transcriptional mechanism, when the bacteria is exposed to different environmental stimuli.