Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates.

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein N-acyltransferase (Lnt) catalyzes the transfer of the sn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an activesite mutant of Escherichia coli Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbonnitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by E. coli Lnt.lipoprotein biosynthesis | apolipoprotein N-acyltransferase | crystal structure | enzyme mechanism T he essential outer membrane (OM) of Gram-negative bacteria has a distinct composition, containing a diverse set of phospholipids, lipopolysaccharides, OM proteins, and lipoproteins (1). Escherichia coli expresses at least 90 lipoproteins that play critical roles in multiple cellular processes ranging from OM biogenesis to cell division and virulence (2). A common feature of these proteins is an N-terminal N-acyl, S-diacylglyceryl cysteine, which ensures their proper localization (3, 4). Before triacylation, preprolipoproteins are translated in the cytoplasm and transferred across the inner bacterial membrane via the Sec or Tat pathway machineries (5). These nascent preprolipoproteins have an N-terminal hydrophobic signal peptide that contains a conserved lipobox motif with the consensus sequence [LVI] -3 [ASTVI][GAS]C +1 , which directs them to the lipoprotein biosynthetic pathway (6-8). The maturation of lipoproteins is then catalyzed by the consecutive action of three essential integral membrane proteins (Fig. 1A). In the first step of this pathway, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) catalyzes the covalent attachment of the sn-1,2-diacylglyceryl group from phosphatidylglycerol to the sulfydryl group of the ...

Osteopontin (OPN), a phosphorylated glycoprotein that binds to an integrin-binding motif, has been shown to regulate nitric oxide (NO) production via inhibition of induced NO synthase (iNOS) synthesis. In the transplanted islets, iNOS and toxic amounts of NO are produced as a result of islets infiltration with inflammatory cells and production of proinflammatory cytokines. Here, we demonstrate that addition of OPN before IL-1beta in freshly isolated rat islets improved their glucose stimulated insulin secretion dose-dependently and inhibited IL-1beta-induced NO production in an arginine-glycine-aspartate-dependent manner. Transient transfection of OPN gene in RINm5F beta-cells fully prevented the toxic effect of IL-1beta at concentrations that reduced the viability by 50% over 3 d. OPN prevention of IL-1beta-induced toxicity was accompanied by inhibited transcription of iNOS by 80%, resulting in 50% decreased formation of the toxic NO. In OPN-transfected cells, the IL-1beta-induced nuclear factor-kappaB activity was significantly reduced. Islets exposed to IL-1beta revealed a naturally occurring early up-regulated OPN transcription. OPN promoter activity was increased in the presence of IL-1beta, IL-1beta-induced NO, and an inducer of NO synthesis. These data suggest the presence of a cross talk between the IL-1beta and OPN pathways and a unique trans-regulatory mechanism in which IL-1beta-induced NO synthesis feedback regulates itself through up-regulation of OPN gene transcription. Our data also suggest that influencing OPN expression represents an approach for affecting cytokine-induced signal transduction to prevent or reduce activation of the cascade of downstream devastating effects after islet transplantation.

De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5=-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuXguaB-guaA deletion in vivo. In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCEDe novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus. We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.