Pathogenic bacteria experience pervasive RNA polymerase backtracking during infection

Abstract

ABSTRACT Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms, including some that cause DNA damage. How these host defense mechanisms impact molecular transactions along the bacterial chromosome during infection is unclear. This is partially due to the lack of a method that can detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that genes experiencing backtracking are downregulated. We observed that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis and for regulation of gene expression during infection. Altogether, our findings have important implications for both efficient transcription and stalled RNAP-driven mutagenesis, which can promote antimicrobial resistance and/or hypervirulence. These results suggest that the host can accelerate the evolution of bacterial pathogens, potentially at their own expense. IMPORTANCE Eukaryotic hosts have defense mechanisms that may disrupt molecular transactions along the pathogen’s chromosome through excessive DNA damage. Given that DNA damage stalls RNA polymerase (RNAP) thereby increasing mutagenesis, investigating how host defense mechanisms impact the movement of the transcription machinery on the pathogen chromosome is crucial. Using a new methodology we developed, we elucidated the dynamics of RNAP movement and association with the chromosome in the pathogenic bacterium Salmonella enterica during infection. We found that dynamics of RNAP movement on the chromosome change significantly during infection genome-wide, including at regions that encode for key virulence genes. In particular, we found that there is pervasive RNAP backtracking on the bacterial chromosome during infections and that anti-backtracking factors are critical for pathogenesis. Altogether, our results suggest that, interestingly, the host environment can promote the development of antimicrobial resistance and hypervirulence as stalled RNAPs can accelerate evolution through increased mutagenesis.