Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

Abstract

When nucleotide-binding oligomerization domain-like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN +/− mice were more responsive to inflammasome activation than those from GLMN +/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.

Regulation of inflammasomes by ubiquitination

“…Shigella species target GLMN by translocation of an E3 ligase (invasion plasmid antigen H7.8) into host cells; IpaH7.8 ubiquitinates GLMN, resulting in its degradation at the proteasome and relieving its inhibition of an unidentified Cullin ring ligase. 45 This study provides indirect evidence that activity of a Cullin ring ligase may be important for NLRP3 activation, and proposes that ubiquitination provides a non-degradative, activating stimulus for the NLRP3 inflammasome.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…[103][104][105] Another NEL effector, IpaH7.8, possesses E3 ligase activity in vitro and targets an inhibitor of Cullin-based RING E3 ligase named GLMN for degradation, which augments inflammasome activation. 106 In addition, OspG and OspI, belonging to a different subsets of S. flexneri effectors, have recently been identified to suppress NF-κB activation by disrupting the ubiquitin system. Mammalian serine/threonine kinase imitator OspG binds to many ubiquitinated E2 proteins, including UbcH5 and Ubch7, in host cells, thereby enhancing its own kinase activity and preventing the ubiquitination and degradation of phosphorylated IκBα.…”

Role of Virulence Factors on Host Inflammatory Response Induced by Diarrheagenic Escherichia Coli pathotypes

“…The mechanism by which IpaH7.8 induced macrophage cell death is via IpaH7.8 E3 ligasemediated proteasome-dependent GLMN degradation in a cell model of BMDMs and peritoneal macrophages isolated from BALB/c mice. GLMN is polyubiquitinated in the presence of IpaH7.8 [139]. In this context, Wang et al, showed that the production and secretion of IpaH4.5 by Shigella interacts with p65 inhibiting the transcriptional activity of NF-κB through the NTR domain of IpaH4.5 and the p65 region spanning from amino acids 1-190.…”