Gut microbiome analysis in piglet models infected with Escherchia coli K88: the role of charcoal and dietary crude protein supplemented with probiotic Escherchia coli strains UM2 and UM7.

Entrotoxigenic Escherichia coli (ETEC) K88 is a causative agent of post-weaning diarrhea (PWD) in early-weaned pigs. This study investigated the efficacy of two alternative diets, charcoal (0.1, 0.5, 1, and 2%) and a low crude protein (CP) diet (17%) supplemented with probiotic E. coli strains (UM2 and UM7), against PWD infection in ETEC K88 challenged piglets. The present study found that charcoal had no effect on the challenged piglets’ performance, ileal and colonic microbiota or their fermentation end products. There was, however, a correlation between charcoal dosage and fecal consistency score. Charcoal reduced the ileal mucosal attached ETEC K88. Feeding a low-CP diet resulted in a lower ileal ammonia concentration. The low-CP diet reduced the E. coli populations in the ileal digesta as well as lowered mRNA expression of the IL-1ß. We concluded that the use of both 1-2% charcoal diet and a low-CP diet supplemented with probiotic E. coli strains were effective in reducing the incidence and severity of PWD infection.October 201

Preferential production of G-CSF by a protein-like Lactobacillus rhamnosus GR-1 secretory factor through activating TLR2-dependent signaling events without activation of JNKs

Different species and strains of probiotic bacteria confer distinct immunological responses on immune cells. Lactobacillus rhamnosus GR-1 (GR-1) is a probiotic bacterial strain found in both the intestinal and urogenital tracts, and has immunomodulatory effects on several cell types including macrophages. However, detailed immunological responses and the signaling mechanism involved in the response are largely unknown

p62/SQSTM1 enhances NOD2-mediated signaling and cytokine production through stabilizing NOD2 oligomers (P4189)

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-kB activation and subsequent production of cytokines (IL-1b and TNF-a). 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.</jats:p

Additional file 1: Table S1. of Preferential production of G-CSF by a protein-like Lactobacillus rhamnosus GR-1 secretory factor through activating TLR2-dependent signaling events without activation of JNKs

Expression of cytokines and chemokines in immortalized BMDMs treated with GR-1 and TLR ligands. (DOC 105 kb

p62 stabilizes gMDP-induced NOD2 oligomers.

<p><b>A</b>. HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p<0.05 (Student t-test). <b>B</b>. HEK293T cells were transiently transfected with Myc-NOD2, HA-NOD2, and scramble (si-Scramble) or p62-targeting (si-Scramble) small interference RNAs. Myc-NOD2 was immunoprecipitated with anti-Myc antibodies and immunoblots were performed against HA or Myc. Immunoblots for total lysates against HA and p62 were performed for HA-NOD2 and endogenous p62 inputs (3^rd and 4^th lanes, respectively). <b>C</b>. HEK293T cells were transfected with Myc-NOD2 at 16 h post-transfection with scrambled siRNA or p62-siRNA. After 24 h, cells were further cultured without or with gMDP (5 µg/ml) for 4 h and cell extracts were loaded onto the Superdex™ 200 column. Fractions were analyzed by immunoblot using Myc antibody for estimation of Myc-NOD2 oligomerization (upper panel). Immunoblot for p38 was used as a control. Myc-NOD2 and p38 immunoreactivities were analyzed using NIH ImageJ program (bottom panel).</p

p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

<div><p>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.</p> </div

The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62.

<p><b>A</b>. NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. <b>B</b>. HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). <b>C</b>. Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.</p