A type III effector protease NleC from enteropathogenic Escherichia coli targets NF‐κB for degradation

Jaclyn S Pearson,Elizabeth L Hartland,Patrice Riedmaier,Olivier Marchès,Gad Frankel

doi:10.1111/j.1365-2958.2011.07568.x

Jaclyn S Pearson, Elizabeth L Hartland + Show 3 more

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https://doi.org/10.1111/j.1365-2958.2011.07568.x

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Abstract

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP–NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His6-NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation.

Highlights

Bacterial pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) utilize a type III secretion system (T3SS) to translocate multiple effector proteins into infected cells (Coburn et al, 2007)
The T3SS of EPEC and EHEC is encoded within the locus of enterocyte effacement (LEE) pathogenicity island which is essential for the ability of the pathogens to cause attaching and effacing (A/E) lesions (McDaniel and Kaper, 1997)
We observed that cells expressing GFP–NleC showed significantly weaker p65 staining than cells expressing GFP, GFP–NleE, GFP–NleD, GFP–NleF, GFP–NleG, suggesting that p65 was undergoing degradation (Fig. 1)

Summary

Introduction

Bacterial pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) utilize a type III secretion system (T3SS) to translocate multiple effector proteins into infected cells (Coburn et al, 2007). Intimate attachment results from a high-affinity interaction between the outer membrane adhesin, intimin and its receptor, Tir, which is translocated into the host cell membrane by the LEE-encoded T3SS (Kenny et al, 1997; Hartland et al, 1999; Luo et al, 2000). The NleG type III effectors are a newly described family of U-Box E3 ubiquitin ligases Their various targets are unknown, the NleG proteins are likely to direct the turnover of host cell proteins and/or other type III effectors by the cell proteasome through the host cell ubiquitination pathway (Wu et al, 2010).

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