Dataset Information

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-?B p65 Cleavage.

ABSTRACT: Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-?B signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which-in contrast to the direct cytoplasmic injection of T3SS effector proteins-are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida (Phdp). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-?B pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1?. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

SUBMITTER: Stolle AS

PROVIDER: S-EPMC5390045 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic <i>Escherichia coli</i> Leads to NF-κB p65 Cleavage.

Stolle Anne-Sophie AS Norkowski Stefanie S Körner Britta B Schmitz Jürgen J Lüken Lena L Frankenberg Maj M Rüter Christian C Schmidt M Alexander MA

Frontiers in cellular and infection microbiology 20170413

Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic <i>Escherichia coli</i> (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the ...[more]

PMID: 28451521

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Project description: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.

Project description:Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-?B) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-?B signaling by cleaving NF-?B Rel subunits. However, it remains elusive how NleC recognizes NF-?B Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹?³? fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-?B target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹?³? fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-?B that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-?B-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-?B signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Dataset Information

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-?B p65 Cleavage.

Publications

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic <i>Escherichia coli</i> Leads to NF-κB p65 Cleavage.

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