Caracterização do internalizador de peptídeos Opp em Corynebacterium pseudotuberculosis e o estudo do seu papel na virulência e patogenicidade dessa bactéria

Abstract

Corynebacterium pseudotuberculosis is a Gram-positive, facultative intracellular parasite, which causes caseous lymphadenitis (CL), a chronic infectious disease in small ruminants, generating great economic losses for sheep and goat producers. Its worldwide occurrence, economic gravity and scarce information stimulate studies on the molecular basis of C. pseudotuberculosis virulence mechanisms. Among molecular factors associated to virulence of many pathogenic bacteria, the oligopeptide importers (Opp), which are multi sub-unitary protein complexes belonging to the ABC transporters family, can be highlighted. These Opp are located in the membrane and their main function is to capture peptides from the extracellular environment to serve as carbon and nitrogen source. In addition to their nutritional role, these peptides may also be used by Gram-positive bacteria as signaling molecules in a process of intercellular communication, which allows bacteria to coordinate the expression of certain genes in a population scale. The control of several cellular processes has been linked to the signal peptides communication system, which include: sporulation, conjugation, and virulence. In order to study the role of the peptide transporter (Opp) in the C. pseudotuberculosis bacterium, a mutant strain for the Opp transporter was constructed using a suicide plasmid. This strain was grown in the presence of the toxic peptide glutathione (GSH) with concentrations of 5mM and 10 mM in order to confirm the lack of ability of the mutant strain to internalize peptides. However, the mutant strain was sensitive to the toxic effects of GSH, showing that this peptide could be internalized by the bacteria through a different mechanism than Opp transport system or that it may not need to be internalized to be toxic to bacteria. In addition, a comparative proteomic analysis between the extracellular proteins from wild and mutant strains using 2-DE, made it possible to generate protein maps showing the presence of 11 unique spots for the wild strain and 17 unique spots for the mutant strain. This result indicates that the transporter Opp may be related to some regulation process of gene expression in C. pseudotuberculosis. Finally, to verify if the peptide transporter of C. pseudotuberculosis has any relation to its virulence and pathogenicity, in vitro tests, adhesion and colonization of macrophages in vivo and infection assays of BALB/c mouse were carried out. The results of these tests showed that although the mutant strain has a reduced capacity to adhere and infect macrophages in culture during the first hour of experimentation, both strains showed the same potential to cause death, injury, and to colonize the spleens of infected mice. As such, we can conclude for this study that the oppD gene mutation did probably not affect the virulence of the mutant strain of C. pseudotuberculosis and that the individual protein spots of each strain should be sequenced to infer to which cellular processes these proteins are related.