Construção de um mutante para o gene sigH codificador do fator sigma alternativo sH e análise do papel desse fator na resposta de Corynebacterium pseudotuberculosis a diferentes condições de estresse ambiental

Abstract

Corynebacterium pseudotuberculosis is a Gram-positive bacterium, which causes caseous lymphadenitis in sheep and goats. In order to cause a successful infection, this facultative intracellular pathogen needs to respond efficiently to changes in the extracellular environment, thus, being able to survive to the harsh conditions encountered in its hostorganism. One of the main mechanisms used by pathogenic bacteria to achieve this goal is the transient activation of specific genes in response to extracellular stresses by the action of the alternative sigma (s) factors of RNA polymerase. After the sequencing of the genome of C. pseudotuberculosis by Rede Genoma de Minas Gerais and Rede Paraense de Genômica e Proteômica, it was identified seven alternative sigma factors in this bacterium, namely the sigma factors B, C, D, E, H, K and M. Among these, the sH factor stands out since it has been linked to virulence in Mycobacterium tuberculosis, a bacterium that is closely related to C. pseudotuberculosis, and to the regulation of important components of M. tuberculosiss as well as of C. glutamicums response to heat shock and oxidative stress. In this context, our research group has been studying the role of the alternative sigma factors in C. pseudotuberculosiss response to different stress conditions in vitro and in its virulence. In this work, therefore, we constructed a strain of C. pseudotuberculosis deficient for the sH factor, using a plasmid that does not replicate in this species, and evaluated its resistance to acid, heat, oxidative and osmotic stresses in vitro by the comparison of the growth curves of the mutant strain and of the wild-type strain when they were exposed to these stress conditions. There were no significant differences in the susceptibility of the mutant strain for acid, heat and oxidative stresses in relation to the susceptibility of wild-type strain. However,it was found that this strain is significantly more susceptible to osmotic stress than the wildtype strain, showing that the sH factor most probably plays an important role in C. pseudotuberculosiss response to this stress condition and, possibly, in this bacteriums survival within macrophages and in its virulence