Abstract
Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H2O2) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD -deficient mutants to study the ability of R. equi to survive exposure to H2O2 in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H2O2. Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H2O2 assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (±122.6) in response to exposure to 50 mM of H2O2 added in the stationary phase, and 3.11 times (±0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H2O2 resistance capability of R. equi.
Highlights
Rhodococcus equi is a Gram-positive, facultative intracellular pathogen affecting foals up to six months of age
R. equi pathogenicity is closely linked to the bacterium’s ability to survive and replicate in the lung macrophages of infected foals [6], where bacteria are generally killed by the combined action of a low pH (4.0–5.0), hydrolytic enzymes and the production of reactive oxygen metabolites such as hydrogen peroxide, H2O2 [7]
We studied the role of these proteins in H2O2 resistance and in the ability of R. equi to survive within mouse peritoneal macrophages
Summary
Rhodococcus equi is a Gram-positive, facultative intracellular pathogen affecting foals up to six months of age. R. equi pathogenicity is closely linked to the bacterium’s ability to survive and replicate in the lung macrophages of infected foals [6], where bacteria are generally killed by the combined action of a low pH (4.0–5.0), hydrolytic enzymes (including proteases, lipases, DNAses and RNAses) and the production of reactive oxygen metabolites such as hydrogen peroxide, H2O2 [7]. R. equi has been characterized as being able to survive and multiply within a membrane-bound vacuole inside macrophages. This capability is conferred by the presence of a 80–90 kb virulence plasmid which inhibits i) the maturation of phagosomes, leading to the absence of phagosome acidification and prevents ii) the fusion of phagosomes with the lysosomes containing many hydrolytic enzymes [7]. Four genes in particular display homology with catalases [10], which are enzymes responsible for detoxifying H2O2 [11]
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