Shigella IpaH zero seven two two E three Ubiquitin Ligase Effector Targets TRAF two to Inhibit PKC-NF-KB Activity in Invaded Epithelial Cells

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Shigella IpaH zero seven two two E three Ubiquitin Ligase Effector Targets TRAF two to Inhibit PKC-NF-KB Activity in Invaded Epithelial Cells

Abstract

NF-KB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-KB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-KB signaling pathway. Here, we show the inhibition of the NF-KB activation by the delivery of the IpaH E three ubiquitin ligase family member IpaH zero seven two two using Shigella's type three secretion system. IpaH zero seven two two dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-KB by ubiquitinating TRAF two, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

Introduction

The intestinal epithelium deploys multiple sensors and defense systems against microbial intrusion. Microbial components and infection-associated cellular damage are recognized as pathogen-associated molecular patterns and as danger-associated molecular patterns, respectively. Pattern recognition receptors recognize pathogen-associated molecular patterns and danger-associated molecular patterns, thus activating the immune system to clear the bacteria and initiate the repair of the injured epithelial lining. Nevertheless, many bacterial pathogens such as Shigella, Salmonella, Yersinia, enteropathogenic Escherichia coli, and enterohemorrhagic E. coli, are able to efficiently colonize the intestinal epithelium by utilizing highly evolved mechanisms to counteract host innate defense mechanisms. Previous studies have reported that enteric bacterial pathogens possess distinctive mechanisms to attenuate host inflammatory responses, which is a prerequisite for promoting intracellular and extracellular bacterial survival. For example, during the invasion of the intestinal epithelium by Shigella, the Toll-like receptors and Nod-like receptors are the Pattern recognition receptors that recognize pathogen-associated molecular patterns and danger-associated molecular patterns. These receptors stimulate host inflammatory signaling pathways, including nuclear factor KB and mitogen activated protein kinases, which culminate in the expression of proinflammatory chemokines and cytokines. Shigella counteract innate immune responses by delivering effector molecules using its type three secretion system. The targeting molecules and mechanisms of inhibition of the NF-KB pathway are specific to each of the effectors, and Shigella are known to deliver OspG, OspI, OspZ, and IpaH nine point eight, to efficiently attenuate NF-KB activation thus allowing replication within the intestinal epithelium.

IpaH nine point eight belongs to IpaH E three ubiquitin ligase family and it is also called novel E three ligase. The ipaH genes, which are originally identified in Shigella large virulence plasmid, are conserved by Gram-negative bacterial pathogens, including Shigella, Salmonella, Yersinia, Edwardshiella ictluri, Bradyrhizobium japonica, Rhisobium sp. strain NGR two three four, Pseudomonas putida, P. entomophila, P. fluorescens, and P. syringae. The IpaH family proteins share common structural and functional properties; they contain an N-terminus leucine rich repeat and a highly conserved C-terminal region. Within the conserved C-terminal region there is a Cys residue that is critical for its E three ubiquitin ligase activity. Each of the IpaH family effectors, including IpaH nine point eight (Shigella), SlrP (Salmonella), SspH one (Salmonella), SspH two (Salmonella), YopM (Yersinia), Y four f R (Rizobium), and BIpM (Yersinia), has been shown to target specific host proteins in a variety of cell types; some of them act as effectors that attenuate host inflammatory responses, while others modulate host defense responses in plants. The existence of multiple effectors with E three ligase activity suggests that a divergent array of E three ligases is required for promoting bacterial infection and antagonizing host innate defense responses.

The Shigella flexneri strain, YSH six thousand, has three ipaH genes (ipaH nine point eight, ipaH seven point eight, and ipaH four point five) on its large virulence plasmid and seven ipaH genes on its chromosome (ipaH zero seven two two, ipaH zero eight eight seven,

Author Summary

Results

IpaH zero seven two two selectively inhibits phorbol myristate acetate-induced NF-KB activation

Ectopically-expressed IpaH zero seven two two localizes to the cell membrane

Membrane rupture of epithelial cells by Shigella invasion triggers PKC-NF-KB activation

IpaH0722 inhibits the protein kinase C-NF-KB pathway without targeting the CBM complex

IpaH zero seven two two preferentially inhibits TRAF two-mediated NF-KB activation

IpaH zero seven two two targets TRAF two for ubiquitination and proteasomal degradation

Discussion

Materials and Methods

Materials

Cell culture

Bacterial infection

Luciferase reporter assays

Expression and purification of recombinant proteins

Pull-down assay

Immunoprecipitation

In vitro ubiquitination assays

Cycloheximide chase assay

RNAi

Murine pneumonia model

Ethics statement

Statistical analysis

Supporting Information

Author Contributions

Overview

The study investigates how Shigella's effector protein IpaH zero seven two two interferes with NF-KB activation, crucial for innate immune responses, by targeting TRAF2 for ubiquitination and degradation, thereby enhancing the bacterium's survival within host cells.

Key Points

1IpaH zero seven two two inhibits NF-KB activation by ubiquitinating TRAF2
2The effector protein targets the PKC-NF-KB pathway during Shigella invasion
3The study used multiple cell lines and mouse models to assess IpaH's role
4E3 ubiquitin ligase activity of IpaH zero seven two two is essential for its function
5Membrane localization of IpaH zero seven two two is critical for its effectiveness in inhibiting NF-KB.

Details

Authors: Hiroshi Ashida, Hiroyasu Nakano, Chihiro Sasakawa
Category: Biology and Natural Sciences

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