Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi

Abstract

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives.

A Novel Screening Strategy Reveals ROS-Generating Antimicrobials That Act Synergistically against the Intracellular Veterinary Pathogen Rhodococcus equi

“…Rhodococcus equi 103S + was kindly provided by Dr. Jesús Navas from the University of Cantabria (Spain), which was considered the wild type strain in all assays. R. equi ∆mrx1∆mrx2∆mrx3 and R. equi ∆mrx1∆mrx2∆mrx3 expressing mrx1-roGFP2 are derivative strains of R. equi 103S + [17]. All reagents were procured from Sigma-Aldrich, unless otherwise stated.…”

“…The biosensor Mrx1-roGFP2 [17] was used to evaluate the intracellular Redox potential of R. equi 103S + during infection. The redox status of Mrx1-roGFP2 was measured as described previously [17].…”

“…The biosensor Mrx1-roGFP2 [17] was used to evaluate the intracellular Redox potential of R. equi 103S + during infection. The redox status of Mrx1-roGFP2 was measured as described previously [17]. Briefly, pellets of exponentially growing R. equi ∆mrx1∆mrx2∆mrx3 expressing mrx1-roGFP2 [17] were resuspended in Phosphate-Buffered Saline (PBS) with different concentrations of antibiotics, treated with 10% N-Ethylmaleimide (NEM) to block free thiol groups, and fixed with 70% ethanol on poly-l-lysine-treated microscope slides.…”

“…Therefore, the antioxidant systems of bacterial pathogens could be important to counteract antibiotic ROS production. We recently demonstrated that mycoredoxins are key factors for the maintenance of R. equi's redox homeostasis [17]. In particular, a R. equi mutant strain lacking three mycoredoxin-encoding genes (R. equi ∆mrx1∆mrx2∆mrx3) shows high susceptibility to oxidative stressing agents, including hydrogen peroxide, sodium hypochlorite, or nitric oxide [17].…”

“…We recently demonstrated that mycoredoxins are key factors for the maintenance of R. equi's redox homeostasis [17]. In particular, a R. equi mutant strain lacking three mycoredoxin-encoding genes (R. equi ∆mrx1∆mrx2∆mrx3) shows high susceptibility to oxidative stressing agents, including hydrogen peroxide, sodium hypochlorite, or nitric oxide [17]. Based on this evidence, we used here this triple mrx-null mutant strain of R. equi to screen for novel ROS-generating antimicrobial compounds.…”