The ubiquitin-binding adaptor proteins p62/SQSTM1 and NDP52 are recruited independently to bacteria-associated microdomains to target Salmonella to the autophagy pathway

Cell Biology Program, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.
Autophagy (Impact Factor: 11.75). 03/2011; 7(3):341-5. DOI: 10.4161/auto.7.3.14046
Source: PubMed


Autophagy is an innate immune defense against bacterial invasion. Recent studies show that two adaptor proteins, p62 and NDP52, are required for autophagy of the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium). However, it is not known why two different adaptors are required to target the same bacterial cargo to autophagy. Here we show that both adaptors are recruited to bacteria with similar kinetics, that they are recruited to bacteria independently of each other, and that depletion of either adaptor leads to impairment of antibacterial autophagy. Depletion of both adaptors does not synergistically impair autophagy, indicating they act in the same pathway. Remarkably, we observed that these adaptors do not colocalize, but rather form non-overlapping microdomains surrounding bacteria. We conclude that p62 and NDP52 act cooperatively to drive efficient antibacterial autophagy by targeting the protein complexes they coordinate to distinct micro-domains associated with bacteria.

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Available from: John Brumell, Mar 07, 2014
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    • "At 1 h p.i., ∼20% of the internalized bacteria were detected by LPS antibodies, indicating they were free in the cytosol or had a compromised vacuolar membrane. This is in good agreement with previous reports where the proportion of cytosolic Salmonella at 1 h p.i. was estimated by the recruitment of autophagy proteins such as LC3, NDP52 and p62 [13], [34]. In cells where at least one bacterium was cytosolic (LPS-positive), we scored the total number of bacteria that had entered that cell. "
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    ABSTRACT: Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "Interestingly, it has been shown that the two adaptor proteins, p62 and NDP52, are recruited at the same time (i.e. 60–90 min post infection) to bacteria-associated microdomains independently of each other and that both are required for antibacterial autophagy of Salmonella enterica (Cemma et al., 2011).Of note, double knockdown of both effectors do not have an additive effect on autophagy impairment indicating that they function in the same pathway and are not redundant (Cemma et al., 2011).Other pathogens that occasionally invade the cytoplasm such as Streptococcus pyogenes are also restricted by NDP52 and TBK1. Recently, the mechanism by which TBK1 restricts bacterial proliferation has been uncovered. "
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    ABSTRACT: Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.
    Full-text · Article · Sep 2013 · Frontiers in Cellular and Infection Microbiology
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    • "Of the more than 30 different proteins involved in autophagy, p62 (also known as sequestosome-1), is an adaptor protein which sequesters poly-ubiquitinated proteins [28] and Salmonella-containing vacuoles [29] to autophagy through interacting with microtubule-associated protein 1 light chain 3 (LC3). In addition to these catabolic roles, p62 has also been shown to regulate various signaling events. "
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    ABSTRACT: NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1β and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.
    Full-text · Article · Feb 2013 · PLoS ONE
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