The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

Constitutive Activation of PrfA Tilts the Balance of Listeria monocytogenes Fitness Towards Life within the Host versus Environmental Survival

PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells

A Role for SKN-1/Nrf in Pathogen Resistance and Immunosenescence in Caenorhabditis elegans

A proper immune response ensures survival in a hostile environment and promotes longevity. Recent evidence indicates that innate immunity, beyond antimicrobial effectors, also relies on host-defensive mechanisms. The Caenorhabditis elegans transcription factor SKN-1 regulates xenobiotic and oxidative stress responses and contributes to longevity, however, its role in immune defense is unknown. Here we show that SKN-1 is required for C. elegans pathogen resistance against both Gram-negative Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis bacteria. Exposure to P. aeruginosa leads to SKN-1 accumulation in intestinal nuclei and transcriptional activation of two SKN-1 target genes, gcs-1 and gst-4. Both the Toll/IL-1 Receptor domain protein TIR-1 and the p38 MAPK PMK-1 are required for SKN-1 activation by PA14 exposure. We demonstrate an early onset of immunosenescence with a concomitant age-dependent decline in SKN-1-dependent target gene activation, and a requirement of SKN-1 to enhance pathogen resistance in response to longevity-promoting interventions, such as reduced insulin/IGF-like signaling and preconditioning H2O2 treatment. Finally, we find that wdr-23(RNAi)-mediated constitutive SKN-1 activation results in excessive transcription of target genes, confers oxidative stress tolerance, but impairs pathogen resistance. Our findings identify SKN-1 as a novel regulator of innate immunity, suggests its involvement in immunosenescence and provide an important crosstalk between pathogenic stress signaling and the xenobiotic/oxidative stress response

Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

<p>Abstract</p> <p>Background</p> <p>The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of <it>β</it>-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the <it>β</it>-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs.</p> <p>Results</p> <p>We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on <it>β</it>-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and <it>β</it>-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the <it>β</it>-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a <it>β</it>-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive.</p> <p>Conclusions</p> <p>Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are <it>β</it>-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.</p

TAC102 is a novel component of the mitochondrial genome segregation machinery in trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization

Dissecting cause and effect in host-microbiome interactions using the combined worm-bug model system

High-throughput molecular studies are greatly advancing our knowledge of the human microbiome and its specific role in governing health and disease states. A myriad of ongoing studies aim at identifying links between microbial community disequilibria (dysbiosis) and human diseases. However, due to the inherent complexity and heterogeneity of the human microbiome we need robust experimental models that allow the systematic manipulation of variables to test the multitude of hypotheses arisen from large-scale ‘meta-omic’ projects. The nematode C. elegans combined with bacterial models offers an avenue to dissect cause and effect in host-microbiome interactions. This combined model allows the genetic manipulation of both host and microbial genetics and the use of a variety of tools, to identify pathways affecting host health. A number of recent high impact studies have used C. elegans to identify microbial pathways affecting ageing and longevity, demonstrating the power of the combined C. elegans-bacterial model. Here I will review the current state of the field, what we have learned from using C. elegans to study gut microbiome and host interactions, and the potential of using this model system in the future

Gut mucosal DAMPs in IBD: From mechanisms to therapeutic implications

Endogenous damage-associated molecular patterns (DAMPs) are released during tissue damage and have increasingly recognized roles in the etiology of many human diseases. The inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), are immune-mediated conditions where high levels of DAMPs are observed. DAMPs such as calprotectin (S100A8/9) have an established clinical role as a biomarker in IBD. In this review, we use IBD as an archetypal common chronic inflammatory disease to focus on the conceptual and evidential importance of DAMPs in pathogenesis and why DAMPs represent an entirely new class of targets for clinical translation. </p

Biokinetics and assessment of intake of thorium dioxide

International audienc

A prospective study of Helicobacter pylori seropositivity and the risk for future myocardial infarction among socioeconomically similar U.S. men.

BACKGROUND: The role of Helicobacter pylori as a determinant of cardiovascular disease is controversial. OBJECTIVE: To determine whether previous exposure to H. pylori is associated with an increased risk for myocardial infarction. DESIGN: Prospective case-control study. SETTING: Physicians' Health Study. PARTICIPANTS: Initially healthy U.S. men. MEASUREMENTS: Titers of IgG antibody against H. pylori and several inflammatory markers were measured in baseline blood samples obtained from 445 men who subsequently had a myocardial infarction (case-patients) and 445 men matched for age and smoking status who remained free of vascular disease (controls) during a mean follow-up of 8.9 years. RESULTS: Baseline seropositivity was similar among case-patients and controls (43.4% vs. 44.3%; rate ratio, 0.96 [95% CI, 0.7 to 1.3]). Minimal evidence of association was found between magnitude of seropositivity and subsequent risk and between seropositivity and levels of the inflammatory biomarkers. CONCLUSION: In a socioeconomically homogeneous population, we found limited evidence of association between H. pylori exposure and risk for future myocardial infarction