c Salmonella enterica serovar Typhimurium is a model organism used to explore the virulence strategies underlying Salmonella pathogenesis. Although intestinal mucus is the first line of defense in the intestine, its role in protection against Salmonella is still unclear. The intestinal mucus layer is composed primarily of the Muc2 mucin, a heavily O-glycosylated glycoprotein. The core 3-derived O-glycans of Muc2 are synthesized by core 3 ␤1,3-N-acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce Muc2 but display a thinner intestinal mucus barrier. We began our investigations by comparing Salmonella-induced colitis and mucus dynamics in Muc2-deficient (Muc2 ؊/؊ ) mice, C3GnT ؊/؊ mice, and wild-type C57BL/6 (WT) mice. Salmonella infection led to increases in luminal Muc2 secretion in WT and C3GnT ؊/؊ mice. When Muc2 ؊/؊ mice were infected with Salmonella, they showed dramatic susceptibility to infection, carrying significantly higher cecal and liver pathogen burdens, and developing significantly higher barrier disruption and higher mortality rates, than WT mice. We found that the exaggerated barrier disruption in infected Muc2 ؊/؊ mice was invA dependent. We also tested the susceptibility of C3GnT ؊/؊ mice and found that they carried pathogen burdens similar to those of WT mice but developed exaggerated barrier disruption. Moreover, we found that Muc2 ؊/؊ mice were impaired in intestinal alkaline phosphatase (IAP) expression and lipopolysaccharide (LPS) detoxification activity in their ceca, potentially explaining their high mortality rates during infection. Our data suggest that the intestinal mucus layer (Muc2) and core 3 O-glycosylation play critical roles in controlling Salmonella intestinal burdens and intestinal epithelial barrier function, respectively.

SUMMARYDuring leaf senescence, resources are recycled by redistribution to younger leaves and reproductive organs. Candidate pathways for the regulation of onset and progression of leaf senescence include ubiquitindependent turnover of key proteins. Here, we identified a novel plant U-box E3 ubiquitin ligase that prevents premature senescence in Arabidopsis plants, and named it SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1). Using in vitro ubiquitination assays, we show that SAUL1 has E3 ubiquitin ligase activity. We isolated two alleles of saul1 mutants that show premature senescence under low light conditions. The visible yellowing of leaves is accompanied by reduced chlorophyll content, decreased photochemical efficiency of photosystem II and increased expression of senescence genes. In addition, saul1 mutants exhibit enhanced abscisic acid (ABA) biosynthesis. We show that application of ABA to Arabidopsis is sufficient to trigger leaf senescence, and that this response is abolished in the ABA-insensitive mutants abi1-1 and abi2-1, but enhanced in the ABA-hypersensitive mutant era1-3. We found that increased ABA levels coincide with enhanced activity of Arabidopsis aldehyde oxidase 3 (AAO3) and accumulation of AAO3 protein in saul1 mutants. Using label transfer experiments, we showed that interactions between SAUL1 and AAO3 occur. This suggests that SAUL1 participates in targeting AAO3 for ubiquitin-dependent degradation via the 26S proteasome to prevent premature senescence.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-β is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-β’s role in host defense is unclear. Thus, wildtype and RELM-β gene deficient mice (Retnlb -/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb -/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb -/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-β did not directly increase IEC proliferation, rather RELM-β acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb -/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-β to Retnlb -/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-β and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.

The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana is needed for post-translational activation of aldehyde oxidase and xanthine dehydrogenase by transferring a sulfur atom to the desulfo-molybdenum cofactor of these enzymes. ABA3 is a two-domain protein consisting of an NH 2 -terminal NifS-like cysteine desulfurase domain and a C-terminal domain of yet undescribed function. The NH 2 -terminal domain of ABA3 decomposes L-cysteine to yield elemental sulfur, which subsequently is bound as persulfide to a conserved protein cysteinyl residue within this domain. In vivo, activation of aldehyde oxidase and xanthine dehydrogenase also depends on the function of the C-terminal domain, as can be concluded from the A. thaliana aba3/sir3-3 mutant. sir3-3 plants are strongly reduced in aldehyde oxidase and xanthine dehydrogenase activities due to a substitution of arginine 723 by a lysine within the C-terminal domain of the ABA3 protein. Here we present first evidence for the function of the C-terminal domain and show that molybdenum cofactor is bound to this domain with high affinity. Furthermore, cyanide-treated ABA3 C terminus was shown to release thiocyanate, indicating that the molybdenum cofactor bound to the C-terminal domain is present in the sulfurated form. Co-incubation of partially active aldehyde oxidase and xanthine dehydrogenase with ABA3 C terminus carrying sulfurated molybdenum cofactor resulted in stimulation of aldehyde oxidase and xanthine dehydrogenase activity. The data of this work suggest that the C-terminal domain of ABA3 might act as a scaffold protein where prebound desulfo-molybdenum cofactor is converted into sulfurated cofactor prior to activation of aldehyde oxidase and xanthine dehydrogenase.Molybdenum enzymes catalyze diverse redox reactions in the global carbon, nitrogen, and sulfur cycles (1). In all eukaryotic molybdenum enzymes, the molybdenum atom is coordinated by the dithiolene group of molybdopterin, thus forming the molybdenum cofactor (Moco) 2 (2). According to the coordination chemistry of the molybdenum ligand, eukaryotic molybdenum enzymes can be divided into two groups; Moco with two additional oxo-ligands and a protein-derived cysteinyl sulfur is bound by enzymes of the sulfite oxidase family, whereas enzymes of the xanthine oxidase family have one oxygen, one inorganic sulfur, and one hydroxyl group ligated to the pterin-chelated molybdenum of the active enzyme. Among the four different molybdenum enzymes known in higher plants, sulfite oxidase and nitrate reductase belong to the sulfite oxidase family, whereas aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) are members of the xanthine oxidase family (3). Although it is believed that all of these molybdenum enzymes basically incorporate the same type of Moco, only AO and XDH, but not enzymes of the sulfite oxidase family, require a final enzyme-dependent post-translational modification of the molybdenum center for activity (4). During this modification step, an oxo-ligand of the Moco in inactive AO and XDH...

No abstract