Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli

Summary

Hydrogen peroxide (H₂O₂) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H₂O₂ tolerance remain unclear. The authors of this article published in Antioxidants and led by GCIR member Professor Karl-Heinz Krause, used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genomeof the bacteria Escherichia coli, to identify genes implicated in H₂O₂ tolerance. Their TraDIS analysis identified 10 mutants with fitness defect upon H₂O₂ exposure, among which previously H₂O₂ - associated genes (oxyR, dps, dksA, rpoS, hfq and polA) and other genes with no known association with H₂O₂ tolerance in E. coli (corA, rbsR, nhaA and gpmA). This is the first description of the impact of gpmA, a gene involved in glycolysis, on the susceptibility of E. coli to H₂O₂. Indeed, confirmatory experiments showed that the deletion of gpmA led to a specific hypersensitivity to H₂O₂ comparable to the deletion of the catalase gene katG, the major H₂O₂ scavenger. This hypersensitivity was not due to an alteration of catalase function or expression, and was independent of the carbon source or the presence of oxygen. Transcription of gpmA was upregulated under H₂O₂ exposure, highlighting its role under oxidative stress. In summary, their TraDIS approach identified gpmA as a member of the oxidative stress defense mechanism in E. coli.

This study was granted by Swiss National Science Foundation to Karl-Heinz Krause. The TraDIS work was supported by University of Queensland funding to Ian Henderson.

Full article: https://www.mdpi.com/2076-3921/11/10/2053

Why is it interesting?

Escherichia coli (E. coli) bacteria are a frequent member of the normal microbiota of healthy humans and animals. Most types of E. coli are harmless or cause relatively brief diarrhea. But a few strains can cause food poisoning, urinary tract infection and even septic shock. The burden of diarrheal infections from pathogenic strains of E. coli is immense; in 79 low-income countries alone, more than 200 million episodes of childhood diarrhea due to E. coli and Shigella occur each year. In high-income countries, E. coli is the leading cause of bloodstream infections, accounting for 27% of documented episodes of bacteremia. In addition, E. coli is the leading pathogen of antimicrobial resistance-associated deaths.

Reactive oxygen species (ROS), and more specifically hydrogen peroxide (H₂O₂), have a strong impact on bacterial pathogenesis. H₂O₂ production by Lactobacillus species, a member of the normal microbiota of the gut, the vagina, and the urinary tract, prevents colonization by urinary tract pathogens. H₂O₂ is also produced by phagocytes during the oxidative burst, a necessary step for pathogen clearance. The effect of H₂O₂ on bacteria has been partially studied, but a complete picture of how H₂O₂ affects bacteria and how they respond to it has not been elucidated.

The combination of transposon mutagenesis and high-throughput sequencing is a powerful technique that enables genome-wide interrogation and represents a new standard for global functional genomic studies. In this study, first authored by Myriam Roth, performed transposon-directed insertion site sequencing (TraDIS) to identify E. coli genes involved in tolerance to exogenous H₂O₂ exposure. The results of the study highlighted the role of gpmA, a gene encoding a phosphoglycerate mutase, a glycolysis enzyme, under H₂O₂ exposure. This is the first time that gpmA has been highlighted as a major contributor to E. coli tolerance to H₂O₂, shedding light on the defense mechanism of E. coli to host oxidative attack and linking the bacterial response against oxidative stress to central metabolism.

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