The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Mark Shepherd,Matthew J Sweet,Adi Idris,Robert K Poole,Kate M Peters,Louise V Holyoake,Alastair G Mcewan,Glen C Ulett,Makrina Totsika,Cláudia A Ribeiro,Minh-Duy Phan,Sohinee Sarkar,Maud E S Achard,Dimitrios Ladakis,Mark A Schembri

doi:10.1038/srep35285

Abstract

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Highlights

Macrophages, for example, by bacterial lipopolysaccharide (LPS)[7] and certain inflammatory cytokines, interferon-gamma (IFN-γ)[8]
To assess the sensitivity of Uropathogenic Escherichia coli (UPEC) to nitrosative stress, well diffusion assays were conducted with the nitric oxide (NO) releaser S-nitrosoglutathione (GSNO) using well-characterised UPEC strains associated with symptomatic (CFT073 and EC958) or asymptomatic (83972) infection, and the reference K-12 commensal strain MG1655
To investigate which systems are important for tolerance to nitrosative stress in UPEC, genes encoding cytochrome bd-I, Hmp, NorVW, NrfA and YtfE were deleted in EC958

Summary

Introduction

Macrophages, for example, by bacterial lipopolysaccharide (LPS)[7] and certain inflammatory cytokines, interferon-gamma (IFN-γ)[8]. Gram-negative bacteria can respond to NO-stress in a variety of ways, including detoxification via the flavohemoglobin Hmp[13,14], the flavorubredoxin NorVW15, the nitrite reductase NrfA16, and the recently characterised NO reductase system Hcp/Hcr[17]. E. coli utilises the diiron protein YtfE to repair iron-sulphur clusters damaged by nitrosative stress[18], and possesses the NO-inducible cytochrome bd-I respiratory oxidase that confers resistance to NO19,20. A recent transcriptomic study demonstrates that the cytochrome bd-I complex is highly-expressed in UPEC isolated from patients with UTI24, confirming that microaerobic conditions are encountered during infection, so the contribution of this system to NO tolerance is of particular interest to UPEC survival. Our data indicate that loss of the cytochrome bd-I complex has the greatest impact upon the ability of UPEC to resist the challenges encountered during infection of the urinary tract

Methods

Results

Conclusion