Iron-manganese superoxide dismutases are primarily responsible for antioxidative responses, antibiotic tolerance, and virulence of Salmonella Pullorum

Superoxide dismutase (SOD) is an important component of the bacterial antioxidant system. In Salmonella Pullorum, an avian specialist serovar of Salmonella, four SODs are encoded, of which sod1 and sod3 belong to the Cu/ZnSOD family, and sod2 and sod4 belong to the Fe/MnSOD family. However, their respective potential functions in stress responses have remained unclear. In this study, we found that a (p)ppGpp synthetase mutant of S. Pullorum exhibited reduced antibiotic tolerance and SOD activity. To further understand the roles of SODs in antibiotic tolerance and virulence, four sod single mutants (Δsod1, Δsod2, Δsod3, and Δsod4) and two sod double mutants (Δsod1Δsod3 and Δsod2Δsod4) were constructed. We found that all four SODs contributed to total SOD activity and superoxide tolerance of S. Pullorum. However, this contribution was not uniform, as the Δsod2Δsod4 mutant showed the lowest survival rates under paraquat treatment, and the highest reactive oxygen species accumulation and lowest survival rates under antibiotic treatment, suggesting the key roles of sod2 and sod4 in degrading ROS in these processes. We also demonstrated that Δsod2Δsod4 was more easily eliminated in a chicken macrophage cell, as well as in the spleen and liver of infected chicken hosts, in comparison with the wild-type strain and other sod mutants. The Δsod2Δsod4 mutant was also the only strain which was completely nonlethal to one-day-old chickens. Taken together, our results indicate that, of the four sod genes, the Fe/MnSOD (sod2 and sod4) collectively had the most significant impact on antibiotic tolerance and pathogenicity of S. Pullorum.