BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

The cofilin phosphatase slingshot homolog 1 (SSH1) links NOD1 signaling to actin remodeling.

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.

Cell-based reporter assay to analyze activation of Nod1 and Nod2.

Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of -different bacterial pathogens. Recent studies furthermore support additional functions of these proteins in the control of adaptive immune responses and intestinal homeostasis. Activation of Nod1 and Nod2 by their cognate elicitors triggers inflammatory responses driven by the activation of NF-κB and MAPK pathways. In this chapter, we describe a quick and reliable cell-based assay using a luciferase reporter to measure Nod1- and Nod2-mediated NF-κB activation. The described protocol was successfully applied to analyze the influences of overexpressed proteins and siRNA-mediated knock-down to provide new insights into the regulation of Nod1/2-specific signaling pathways. Furthermore, this method is well suited for downscaling to high-throughput screening applications.

A role for quorum sensing in regulating innate immune responses mediated by Vibrio cholerae outer membrane vesicles (OMVs).

Outer membrane vesicles (OMVs) are released from many Gram-negative bacteria. OMVs interact with and are taken up by human cells. We and others have now showed that OMVs contain peptidoglycan, which is sensed mainly by the pattern-recognition receptor NOD1 in the cytoplasm of host cells. Vibrio cholerae is clinically important as one of the causative agents of severe dehydrating diarrhea in humans. We showed that non-O1 non-O139 V. cholerae (NOVC) strains of V. cholera produce OMVs. Of note, we revealed that NOVC can evade NOD1-mediated immune surveillance by the quorum sensing machinery. Here we review these recent findings and discuss the relevance for our understanding of bacterial infections and innate immune responses.

The yeast ubiquitin-like domain protein Mdy2 is required for microtubule-directed nuclear migration and localizes to cytoplasmic granules in response to heat stress.

MDY2 encodes a ubiquitin-like (UBL)-domain protein necessary for efficient mating in Saccharomyces cerevisiae. Unlike most UBL proteins, Mdy2 is apparently not subject to C-terminal processing and is localized predominantly in the nucleus. Deletion of MDY2 is associated with a five- to seven-fold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. Here, we looked for two potential interacting partners of Mdy2, investigated the function of Mdy2 in nuclear movement, determined the increased heat sensitivity defects of mdy2Δ mutants, and inspected localization of Mdy2. Coprecipitation studies show that Mdy2 associates with α-tubulin and with the microtubule (MT)-associated dynactin subunit p150(Glued)/Nip100. nip100Δ mutants exhibit no defects in nuclear migration or in MT length or orientation during shmooing growth. Deletion of MDY2 display small nuclear migration phenotype during vegetative growth and seems to exacerbate the defects in mitotic nuclear migration seen in the nip100Δ strain. Deletion of MDY2 increased heat sensitivity of the cells and these strains accumulate mitotic nuclear migration defects and shortened MTs under these conditions. GFP-Mdy2 proteins which are localized predominantly in the nucleus at permissive temperature are localized to cytoplasmic foci during heat shock. Colocalization studies revealed that heat stress-induced enrichment of Mdy2 in cytoplasmic foci merged mainly with stress granules marker Pab1. During glucose deprivation a minority of Mdy2 foci overlapped with P-bodies marker Dcp2, while most Mdy2 foci and Pab1 foci overlap. Accordingly, we propose that Mdy2 plays a critical role in the MT-dependent processes of karyogamy and stress response.

Anti-inflammatory arene--chromium complexes acting as specific inhibitors of NOD2 signalling.

Inflammation is a hallmark of microbial infection in mammals and is the result of a pathogen-induced release of inflammatory effectors. In humans a variety of germ-line encoded receptors, so-called pattern-recognition receptors, respond to conserved signatures on invading pathogens, which results in the transcriptional activation of pro-inflammatory responses. Inflammation is often detrimental to the host and leads to tissue damage and/or systemic dysfunctions. Thus, specific inhibitors of these pathways are desirable for medical interventions. Herein we report on the synthesis and use of some chromium-containing compounds (arene--Cr(CO)3 complexes) with a core structure related to anti-inflammatory diterpenes produced by the sea whip Pseudopterogorgia elisabethae. By using cell-based reporter assays we identified complexes with a potent inhibitory activity on tumour necrosis factor (TNF), Toll-like receptor (TLR), and nucleotide binding domain, leucine-rich repeat-containing receptor (NLR) pathways. Moreover, we found one complex to be a specific inhibitor of inflammatory responses mediated by the NLR protein NOD2, a pivotal innate immune receptor involved in bacterial recognition. Synthesis and characterisation of a set of derivatives of this substance revealed structural requirements for NOD2 specificity. Taken together, our studies suggest this type of arene--Cr(CO)3 complex as a potential lead for the development of antiphlogistica and pharmacologically relevant NOD2 inhibitors.