Risk Factors for high serum SeM titers after natural outbreaks of Streptococcus equi equi in horses

Abstract

Rhodococcosis classically caused severe lung affections as suppurative bronchopneumonia and sometimes unusual forms as digestive or bone forms. R. equi is able to survive and multiply in macrophages despite unsuitable conditions, caused notably by reactive oxygen metabolites such as hydrogen peroxide (H2O2) generated during the respiratory burst of phagocytic cells. It was known that R. equi presents a great capacity of resistance to H2O2 exposition independently to the presence of the virulence plasmid. Recently, the analysis of R. equi 103 genome revealed the presence of four potential catalase genes named katA to katD. The aim of this study was to determine the implication of each catalase in R. equi oxidative stress resistance. For that, we used the R. equi 103 plasmid-less strain to construct four catalase deficient mutants, each lacking one catalase gene to obtain DkatA, DkatB, DkatC and DkatD. Survivals of those mutants were analyzed in vitro after exposition to oxidative stress by H2O2 treatment and in vivo by survival assay in mouse peritoneal macrophages. A quantitative RT-PCR analysis of catalase genes expression levels, in R. equi 103 plasmid-less WT, after exposition to H2O2, was also performed. We observed that, after exposition to H2O2 in stationary or exponential phase, survival of DkatA was respectively completely abolished or decreased by two orders of magnitude, compared to the parental strain. Results showed also decrease by three orders of magnitude in DkatC survival after treatment in exponential phase. In vivo experiments in mice macrophages showed that DkatA was the most susceptible mutant and was completely eliminated from macrophages 72h post infection (Figure 1). The others mutants were more susceptible to elimination by macrophages than R. equi 103 WT, but were not completely eliminated 48h and 72h post infection (Figure 1). Quantitative RT-PCR results showed that external H2O2 exposition only increased katA gene expression. After treatment in stationary or exponential phase, katA was respectively overexpressed 367.9 ( 122.6) times and 3.11 ( 0.59) times, compared to untreated conditions. Taken together, our results revealed the main implication of KatA and in a less manner, KatC, in resistance to external H2O2 exposition. The role of the others catalases of R. equi 103 in oxidative stress resistance is not clearly demonstrated in our experimental conditions. In conclusion, this study highlights the main role of katA in response and resistance to external oxidative stress in R. equi 103 plasmid-less starin.