Resumo

Avian Pathogenic Escherichia coli(APEC) are a group of extra-intestinal E. coli that infect poultry and may cause systemic or localized diseases, being one of the most common bacterial illnesses in poultry production. E. coli and Salmonella have two independent pathways for nitrate (NO3-) or nitrite (NO2-) reduction. One localized in the periplasm and the other into the cytoplasm. Nitrite is a central branching point in the nitrogen bacterial metabolism and is used as a macrophage antibacterial cytotoxin as a part of the innate immune response. Nitrite anions are generated by NO3- reduction when it is used as electron acceptor during anaerobic respiration. Nitrite can be also used as electron acceptor employing nitrite reductases that reduce it to ammonia. Intracellular accumulation of nitrite is critical for most cells due spontaneous generation of the cytotoxic nitric oxide radical (NO). Nitrite differently to NO is not diffusible across bacteria. NirC acts as a specific channel to facilitate the transport of NO2- in Salmonella and E. coli for nitrogen metabolism and cytoplasmic detoxification. Furthermore, NirC showed be required for Salmonella Typhimurium pathogenicity by downregulating NO production of host macrophage. Based on a microarray, which revealed overexpression of nirC gene in the APEC strain Sci-07 (ONT:H31), we constructed a Sci-07 nirC-deficient strain and its virulence potential was evaluated. The mutant ΔnirC was constructed using the lambda red knockout system. Virulence evaluation was based on chicken mortality assay; potential of systemic infection by bacteria recovering from organs; bacteria adhesion in avian fibroblast cell line, and intracellular survival of bacteria in avian macrophages cell line. The mortality caused by mutant and wild-type strain (WT) were similar (65%); however ΔnirC provoked gradual mortality rate during the seven days analyzed while the WT caused mortality up to 24 hours-post infection (hpi). Counts of ΔnirC in spleen, lung and liver were higher than the WT after 48 hours post infection (p<0.05) but lower at 24 hpi. Higher intracellular counts of the mutant in macrophages were observed after 16 hpi (p<0.05) but similar counts were observed after 3hpi. The cell adhesion ability of the mutant was a half of the WT strain in presence and absence of mannose. Our work highlighted the role of nirC as a virulence gene confirming previous observation where nirC-deficientSalmonella was unable to survive in macrophage and unable to organs colonization of mice. Nevertheless, to the APEC strain used in this work, nirC seems influence different roles in the pathogenic process. In earlier stage of infection the absence of the gene decreased the virulence of the mutant, and in the later stage of infection the absence of nirC gene improved the fitness of the strain.