Elucidation of the role of stress-induced DNA polymerase II in the pathogenesis of uropathogenic Escherichia coli

Abstract

Introduction: Uropathogenic Escherichia coli (UPEC) strains are responsible for most registered urinary tract infections (UTIs) in humans. Invading bacteria are exposed to harsh conditions resulting from the host's attempts to inactivate them. Inflammatory assault, in turn, triggers bacterial stress responses, which allow the bugs to survive. One of the major global stress responses is the SOS system, induced when DNA damage occurs. Nevertheless, there is virtually no insight which molecular components of SOS are crucial for UPEC colonization. Previously, we revealed that the accessory DNA polymerase IV (dinB), activated as part of the SOS response, may play an important role in bacterial survival strategies. Here, we aimed to investigate the role of DNA polymerase II (Pol II/polB) in the pathogenesis of UPEC. Methods: In the study, UPEC strains (UTI89 and CFT073), the 5637 cell line, and CH3/HeN mice were used. To evaluate the expression level of Pol II in the UPEC strains, SOS was induced by construction of UPEC/lexA(def) strains using the lambda red-mediated genome editing method. Whole transcriptome sequencing (RNA-seq) analysis was used to establish SOS-deregulated genes, including the expression level of polB, in the tested strains. Moreover, Pol II DNA-deficient (ΔpolB) strains were generated using the lambda red-based technique. The constructs were subjected to: (i) mutational frequency analysis in vitro (Rif; Nal); (ii) the gentamicin protection assay (5637 cell line) and UTI mouse model, to determine the ability of the polB mutant to infect and form intracellular colonies within bladder epithelial cells. Data were analyzed using Prism/GraphPad (p<0,05). Results: The data obtained from RNAseq indicate that the Pol II replicase is significantly upregulated in UPEC strains when SOS is activated. It was found that lack of Pol II leads to a slight increase in mutability of UPEC, but does not affect their survival in vitro. Most importantly, it was observed that in vivo, deletion of polB results in a strong decrease in the capability of UPEC to form colonies in the infected bladder epithelial cells. Discussion: Overall, the role of Pol II in maintenance of genome stability was unclear. It was proposed that Pol II can play a back-up role for the main replicase, but its loss under non-stress growth conditions does not affect the cell's phenotype. Our data are the first showing the importance of Pol II and indicate that under stress, UPEC engage and require Pol II to manage genome integrity, without which their viability is threatened. Conclusion: The study highlights the importance of E. coli DNA polymerase II in UPEC pathogenesis and suggests that Pol II may constitute a novel virulence factor of UTIs and therefore, may be considered as a promising treatment target.This study was funded by the National Science Centre, grant 2019/35/B/NZ6/03996.