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

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.

Attaching and Effacing Bacterial Effector NleC Suppresses Epithelial Inflammatory Responses by Inhibiting NF-κB and p38 Mitogen-Activated Protein Kinase Activation

“…Recently several studies have identified NleC as another effector that suppresses inflammatory response through inhibition of NF-B (3,35,38,56). These studies showed that NleC also suppressed IL-8 release and NF-B activation by cultured epithelial cells, specifically through the degradation of the NF-B p65 subunit.…”

“…These studies showed that NleC also suppressed IL-8 release and NF-B activation by cultured epithelial cells, specifically through the degradation of the NF-B p65 subunit. NleC is a zinc protease that cleaves NF-B p65 subunit (3,35,38,56). Curiously, despite the prominent immunosuppressive role attributed to NleC in vitro, no studies have yet examined the potential for NleC to modulate inflammation in vivo.…”

Modulation of host signaling in the inflammatory response by enteropathogenic Escherichia coli virulence proteins

“…28 Other NF-κB components, c-Rel and p50, are also degraded in infected cells. 29 As a result, NleC-mediated proteolysis suppresses NF-κB activation and subsequently impairs the secretion of IL-1β, IL-8 and TNF-α. [28][29][30] The NF-κB component p65, recruited by NleC, is targeted for proteasomal-independent degradation, presumably via a consensus zinc metalloprotease motif.…”

“…29 As a result, NleC-mediated proteolysis suppresses NF-κB activation and subsequently impairs the secretion of IL-1β, IL-8 and TNF-α. [28][29][30] The NF-κB component p65, recruited by NleC, is targeted for proteasomal-independent degradation, presumably via a consensus zinc metalloprotease motif. Early studies suggested that the site of p65 cleavage by NleC lies within its N-terminal Rel homology domain (RHD), which is involved in DNA binding by p65 on adjacent, parallel strands.…”

Enteropathogenic and enterohemorrhagic E. coli: ecology, pathogenesis and evolution

“…For example, NleE is a cysteine methyl transferase that methylates the zinc finger domain of TAB2/3, thereby inhibiting TAB2/3 interaction with ubiquitinated TRAF and blocking the phosphorylation of IκB by the IKK complex and hence the degradation of IκB (Newton et al, 2010;Zhang et al, 2011). NleC and NleD are zinc metalloproteases that cleave the p65 subunit of NF-κB and the MAPKs p38 and JNK, respectively (Baruch et al, 2011;Pearson et al, 2011). NleH has both anti-apoptotic and anti-inflammatory effects acting in part by binding ribosomal protein S3 (Hemrajani et al, 2010;Gao and Hardwidge, 2011).…”