Biblioteca Virtual en Salud

Corynebacterium pseudotuberculosis e um patogeno intracelular de grande relevancia veterinaria que infecta pequenos ruminantes, causando uma enfermidade conhecida como Linfadenite Caseosa (LC). Esta doenca e distribuida mundialmente e esta associada a grandes perdas economicas. Apesar do processo patologico da LC ser bem entendido, existem poucos estudos sobre os determinantes moleculares de virulencia de C. pseudotuberculosis. Neste contexto, nosso grupo de pesquisa tem se dedicado ao estudo das proteinas moduladoras da expressao genica, tais como os fatores sigmas da RNA polimerase bacteriana. A regulacao da expressao genica realizada por fatores sigma alternativos e crucial para a sobrevivencia bacteriana em condicoes ambientais hostis, como a encontrada no ambiente intrafagossomico durante a infeccao. O fator sigma ÐE e o regulador transcricional mais estudado de C. pseudotuberculosis, tendo sido demonstrado seu envolvimento na resposta adaptativa deste microrganismo ao estresse nitrosativo. Entretanto, ainda nao tinha sido realizado um estudo aprofundado acerca da regulacao da expressao do gene sigE, que codifica este fator. Neste trabalho foi realizada a caracterizacao in silico da estrutura da unidade transcricional deste gene e ensaios de RT- PCR na tentativa de confirmar se o gene sigE da linhagem 1002 de C. pseudotuberculosis estaria organizado em operon juntamente com os genes cseE e tatB, genes que codificam respectivamente um possivel regulador negativo do fator sigma E e um componente de um sistema de secrecao alternativo, Twin Arginine Translocase. Alem disso, os niveis de transcricao dos genes sigE, cseE e tatB foram avaliados pela tecnica de RT-qPCR em culturas expostas aos estresses nitrosativo e acido induzidos in vitro a fim de se confirmar envolvimento destes genes na resposta adaptativa especifica a estes estresses. Os resultados obtidos confirmaram que os genes sigE, cseE e tatB constituem um operon que os genes sigE e cseE sao ativados na resposta de C. pseudotuberculosis 1002 aos estresses nitrosativo e acido enquanto que o gene tatB e ativado somente na resposta ao estresse nitrosativo. Tomados juntos, esses dados sugerem que os genes deste operon possam ser regulados diferencialmente, por um mecanismo complexo a nivel transcricional, dependendo do estimulo do ambiente

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