NleC, a Type III Secretion Protease, Compromises NF-κB Activation by Targeting p65/RelA

Abstract

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.

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

“…In addition, NleD can also specifically cleave and inactivate the p65 subunit of NF-κB. 60 EPEC-derived NleC, as mentioned above, was predominantly localized at the site of bacterial attachment beneath the host cell membrane 28 and had an effect on MAPK signaling in addition to NF-κB signaling. 61 Significantly elevated p38 MAPK phosphorylation was detected in NleC-deleted EPEC infection.…”

“…The decreased amount of p65 results in a decrease in the total nuclear entry of active p65. 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.…”

“…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.…”

“…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. 28,30 It has been further shown that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 are needed for the recognition and binding of NleC. 31 In spite of cleavage of a small percentage of p65, modulation of NF-κB-mediated gene expression is greatly amplified by a mechanism by which an NleC cleavage-generated p651-38 fragment interacts with p65 and ribosomal protein S3 (RPS3), which facilitates p65 binding to promoters and gene expression.…”

The Inflammatory Response during Enterohemorrhagic Escherichia coli Infection

“…In fact, NleC degrades the NF-κB subunit, p65, and this function is dependent on the zinc metalloprotease motif, 183 HEIIH (96)(97)(98)(99)102). Degradation is direct, as recombinant His 6 -NleC cleaves both p65 and p50 in epithelial cell lysates (98).…”