Jakob Begun

Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis

ABSTRACT Staphylococcus aureus, an important pathogen of humans and other warm-blooded animals, is also capable of killing the nematode Caenorhabditis elegans. Here, we show that C. elegans organisms that are fed S. aureus die over the course of several days in a process that is correlated with the accumulation of bacteria within the nematode digestive tract. Several S. aureus virulence determinants known or speculated to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr and the global virulence regulator sarA, the alternative sigma factor σB, alpha-hemolysin, and V8 serine protease, are required for full pathogenicity in nematodes. In addition, several defined C. elegans mutants were examined for susceptibility to S. aureus infection. Enhanced susceptibility to S. aureus killing was observed with loss-of-function mutations in the C. elegans genes esp-2/sek-1 and esp-8/nsy-1, which encode components of a conserved p38 MAP kinase signaling pathway involved in nematode defense against multiple pathogens. These results suggest that key aspects of S. aureus pathogenesis have been conserved, irrespective of the host, and that specific C. elegans host factors can alter susceptibility to this gram-positive human pathogen.

IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans

Significance Chronic granulomatous disease (CGD) has an immunodeficiency component and, in addition, an autoinflammatory component in which autophagy and inflammasome activation are linked and amenable to IL-1 blockade. This study provides a rationale to perform clinical trials to investigate the efficacy of blocking IL-1 in CGD colitis and expands the therapeutic potential of IL-1 antagonists to inflammatory diseases with defective autophagy. Patients with chronic granulomatous disease (CGD) have a mutated NADPH complex resulting in defective production of reactive oxygen species; these patients can develop severe colitis and are highly susceptible to invasive fungal infection. In NADPH oxidase-deficient mice, autophagy is defective but inflammasome activation is present despite lack of reactive oxygen species production. However, whether these processes are mutually regulated in CGD and whether defective autophagy is clinically relevant in patients with CGD is unknown. Here, we demonstrate that macrophages from CGD mice and blood monocytes from CGD patients display minimal recruitment of microtubule-associated protein 1 light chain 3 (LC3) to phagosomes. This defect in autophagy results in increased IL-1β release. Blocking IL-1 with the receptor antagonist (anakinra) decreases neutrophil recruitment and T helper 17 responses and protects CGD mice from colitis and also from invasive aspergillosis. In addition to decreased inflammasome activation, anakinra restored autophagy in CGD mice in vivo, with increased Aspergillus-induced LC3 recruitment and increased expression of autophagy genes. Anakinra also increased Aspergillus-induced LC3 recruitment from 23% to 51% (P < 0.01) in vitro in monocytes from CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1–dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade.

Staphylococcal Biofilm Exopolysaccharide Protects against Caenorhabditis elegans Immune Defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host–pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.

Deep Resequencing of GWAS Loci Identifies Rare Variants in CARD9, IL23R and RNF186 That Are Associated with Ulcerative Colitis

Genome-wide association studies and follow-up meta-analyses in Crohns disease (CD) and ulcerative colitis (UC) have recently identified 163 disease-associated loci that meet genome-wide significance for these two inflammatory bowel diseases (IBD). These discoveries have already had a tremendous impact on our understanding of the genetic architecture of these diseases and have directed functional studies that have revealed some of the biological functions that are important to IBD (e.g. autophagy). Nonetheless, these loci can only explain a small proportion of disease variance (∼14% in CD and 7.5% in UC), suggesting that not only are additional loci to be found but that the known loci may contain high effect rare risk variants that have gone undetected by GWAS. To test this, we have used a targeted sequencing approach in 200 UC cases and 150 healthy controls (HC), all of French Canadian descent, to study 55 genes in regions associated with UC. We performed follow-up genotyping of 42 rare non-synonymous variants in independent case-control cohorts (totaling 14,435 UC cases and 20,204 HC). Our results confirmed significant association to rare non-synonymous coding variants in both IL23R and CARD9, previously identified from sequencing of CD loci, as well as identified a novel association in RNF186. With the exception of CARD9 (OR = 0.39), the rare non-synonymous variants identified were of moderate effect (OR = 1.49 for RNF186 and OR = 0.79 for IL23R). RNF186 encodes a protein with a RING domain having predicted E3 ubiquitin-protein ligase activity and two transmembrane domains. Importantly, the disease-coding variant is located in the ubiquitin ligase domain. Finally, our results suggest that rare variants in genes identified by genome-wide association in UC are unlikely to contribute significantly to the overall variance for the disease. Rather, these are expected to help focus functional studies of the corresponding disease loci.

Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model

ABSTRACT Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

DAF-16-Dependent Suppression of Immunity During Reproduction in Caenorhabditis elegans

To further understand how the nematode Caenorhabditis elegans defends itself against pathogen attack, we analyzed enhanced pathogen resistance (epr) mutants obtained from a forward genetic screen. We also examined several well-characterized sterile mutants that exhibit an Epr phenotype. We found that sterility and pathogen resistance are highly correlated and that resistance in both epr and sterile mutants is dependent on DAF-16 activity. Our data indicate that a DAF-16-dependent signaling pathway distinct from previously described pathways is involved in the activation of genes that confer resistance to bacterial pathogens. The timing of DAF-16-dependent gene activation in sterile mutants coincides with the onset of embryonic development in wild-type animals, suggesting that signals from developing embryos normally downregulate the immune response.

High Fat Diets Induce Colonic Epithelial Cell Stress and Inflammation that is Reversed by IL-22

Prolonged high fat diets (HFD) induce low-grade chronic intestinal inflammation in mice, and diets high in saturated fat are a risk factor for the development of human inflammatory bowel diseases. We hypothesized that HFD-induced endoplasmic reticulum (ER)/oxidative stress occur in intestinal secretory goblet cells, triggering inflammatory signaling and reducing synthesis/secretion of proteins that form the protective mucus barrier. In cultured intestinal cells non-esterified long-chain saturated fatty acids directly increased oxidative/ER stress leading to protein misfolding. A prolonged HFD elevated the intestinal inflammatory cytokine signature, alongside compromised mucosal barrier integrity with a decrease in goblet cell differentiation and Muc2, a loss in the tight junction protein, claudin-1 and increased serum endotoxin levels. In Winnie mice, that develop spontaneous colitis, HFD-feeding increased ER stress, further compromised the mucosal barrier and increased the severity of colitis. In obese mice IL-22 reduced ER/oxidative stress and improved the integrity of the mucosal barrier, and reversed microbial changes associated with obesity with an increase in Akkermansia muciniphila. Consistent with epidemiological studies, our experiments suggest that HFDs are likely to impair intestinal barrier function, particularly in early life, which partially involves direct effects of free-fatty acids on intestinal cells, and this can be reversed by IL-22 therapy.

Review article: consensus statements on therapeutic drug monitoring of anti-tumour necrosis factor therapy in inflammatory bowel diseases

Therapeutic drug monitoring (TDM) in inflammatory bowel disease (IBD) patients receiving anti‐tumour necrosis factor (TNF) agents can help optimise outcomes. Consensus statements based on current evidence will help the development of treatment guidelines.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TGs actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.