Lack of RNase HI affects virulence in Salmonella enterica

Bacterial virulence and antibiotic resistance are interconnected global threats that can synergize, multiplying their adverse effects on health and economy. Two proposed strategies to address this dual challenge are non-biocidal inhibition of virulence traits (anti-virulence strategy) and manipulation of eco-evolutionary dynamics in bacterial populations to hamper dissemination of antibiotic resistance (anti-resistance strategy). These strategies require to identify factors involved in bacterial gene expression, fitness and evolution. R-loops strongly influence these traits, as well as fitness of bacteria carrying antibiotic resistance mutation, making the enzyme responsible for R-loop degradation, the RNase HI, a promising target for anti-resistance approaches. Interestingly, the extensive involvement of R-loops in transcription initiation and termination could also make RNase HI a potential target for anti-virulence strategies. Here we explored this possibility by investigating the effects of RNase HI deficiency on traits associated with pathogenicity in the human pathogen Salmonella enterica serovar Typhimurium. We found that lack of RNase HI alters expression of virulence genes at population and single-cell levels, and both ex vivo and during infection of mammalian cells. Moreover, we showed that RNase HI depletion causes defects in phenotypes associated with virulence, such as motility, biofilm formation, and resistance to low pH. Coherently, we observed that the absence of RNase HI reduces Salmonella ’s ability to survive within macrophages. Altogether, our results demonstrate the influence of R-loops on bacterial virulence, suggesting that RNase HI could serve as a target for anti-virulence strategies. This provides a theoretical framework for the development of dual anti-resistance and anti-virulence interventions based on RNase HI targeting.