Avaliação da influência de OxyR na resposta ao estresse e na virulência de Corynebacterium diphtheriae

Abstract

Corynebacterium diphtheriae, the major etiological agent of diphtheria, has been associated to invasive infections, regardless of diphtheria toxin production. This fact suggests that others virulence factors, besides to the diphtheria toxin production, are involved in the pathogenicity of this microorganism. Several mechanisms of survival and dissemination of the microorganism, both in the host and in the environment, contribute to the bacterial infections pathogenesis. Amongst the pathogenic mechanisms, the response of C. diphtheriae to different stress responses still remain misunderstood. The aim of this study was to investigate the role of the OxyR, already described as an important regulator involved in the resistance and virulence of other bacterial species, in the C. diphtheriae pathogenesis. Therefore, the parental strains CDC-E8392 and the mutant (LDCIC-L2), unable to produce OxyR, were compared for gene expression and survival ability under stress conditions. In addition, both strains were compared for their pathogenic potential, through interaction with epithelial cells and macrophages, biofilm formation, interaction with plasma and extracellular matrix proteins assays and in vivo infection to C. elegans nematodes and in Swiss Webster mice. The results indicated that OxyR regulates negatively the catalase expression and response to H2O2. The interruption of expression of OxyR also modulated the expression of cspA and the resistance to oxidative stress generated by ozone. The mutant strain showed a significant reduction in the capacity of binding to fibronectin, collagen and fibrinogen. The influence of OxyR on the virulence properties of C. diphtheriae was also evidenced by ability of the mutant strain to survive in the intracytoplasmic compartment of human epithelial cells and macrophage and to induce a more evident inflammatory process in knee joint in mice. In addition, despite the cellular infection of respiratory cells by C. diphtheriae strains lead to increased production of ROS and RNS, the OxyR– mutant strain induced a significantly higher production of NO than the cells infected with the wild type strain. However, interruption of OxyR did not induce a significant difference in both biofilm formation on hydrophilic (glass) and hydrophobic (polystyrene) abiotic surfaces, in the presence or absence of iron, and in the susceptibility profiles to antimicrobial agents. Futhermore, OxyR seem not to influence the pathogenicity of C. diphtheriae in C. elegans, since there was no significant difference in the nematode colonization and death assays with the strains analyzed, although a greater formation of the bagging effect has been observed in worms infected by the mutant strain. In conclusion, OxyR effects on C. diphtheriae virulence seem not to be solely related to the regulation of both oxidative and cold shock stress response, but are also associated with other clinically relevant processes that allow this pathogen to persist in the host tissues, despite inflammatory and cellular responses.