Browning Kaitlyn R, Merrikh Houra
Vanderbilt University School of Medicine, Department of Biochemistry, Nashville, TN 37232, USA.
bioRxiv. 2023 May 12:2023.05.12.540596. doi: 10.1101/2023.05.12.540596.
Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could 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 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 the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.
致病细菌与其真核宿主处于持续的军备竞赛之中。宿主拥有众多防御机制，这些机制不仅能对抗细菌入侵者，还有可能扰乱细菌染色体上的分子活动。然而，目前尚不清楚宿主在感染过程中如何在分子水平上影响蛋白质与细菌染色体的结合。部分原因是缺乏一种能够在病原体处于宿主细胞内时检测这些事件的方法。我们开发并优化了一种系统，该系统能够在细菌积极感染宿主时（称为PIC-seq）绘制并测量与染色体相关的细菌蛋白质水平。在此，我们以一种致病细菌为模型系统，重点研究了感染过程中RNA聚合酶（RNAP）在染色体上的移动动态及其与染色体的结合情况。利用PIC-seq，我们发现感染过程中全基因组范围内RNAP与染色体的结合模式会发生变化，包括在编码关键毒力基因的区域。重要的是，我们发现宿主感染会显著增加RNAP在细菌染色体上的回溯。RNAP回溯是染色体上RNAP进程最常见的中断形式。有趣的是，我们发现通过抗回溯因子GreA和GreB解决回溯的RNAP对发病机制至关重要，从而揭示了一类新的毒力基因。总之，我们的结果有力地表明，宿主感染通过破坏细菌病原体染色体上的RNAP移动，对转录产生显著影响。增加的回溯事件不仅对高效转录有重要影响，而且对突变率也有影响，因为停滞的RNAP会增加诱变水平。
