Lgt Processing Is an Essential Step in Streptococcus suis Lipoprotein Mediated Innate Immune Activation

Background: Streptococcus suis causes invasive infections in pigs and occasionally in humans. The host innate immune system plays a major role in counteracting S. suis infections. The main components of S. suis able to activate the innate immune system likely include cell wall constituents that may be released during growth or after cell wall integrity loss, however characterization of these components is still limited. Methology/Principal Findings: A concentrated very potent innate immunity activating supernatant of penicillin-treated S. suis was SDS-PAGE fractionated and tested for porcine peripheral blood mononucleated cell (PBMC) stimulating activity using cytokine gene transcript analysis. More than half of the 24 tested fractions increased IL-1b and IL-8 cytokine gene transcript levels in porcine PBMCs. Mass spectrometry of the active fractions indicated 24 proteins including 9 lipoproteins. Genetic inactivation of a putative prolipoprotein diacylglyceryl transferase (Lgt) gene resulted in deficient lipoprotein synthesis as evidenced by palmitate labeling. The Lgt mutant showed strongly reduced activation of porcine PBMCs, indicating that lipoproteins are dominant porcine PBMC activating molecules of S. suis. Conclusion/Significance: This study for the first time identifies and characterizes lipoproteins of S. suis as major activators of the innate immune system of the pig. In addition, we provide evidence that Lgt processing of lipoproteins is required fo

Enrichment of homologs in insignificant BLAST hits by co-complex network alignment

<p>Abstract</p> <p>Background</p> <p>Homology is a crucial concept in comparative genomics. The algorithm probably most widely used for homology detection in comparative genomics, is BLAST. Usually a stringent score cutoff is applied to distinguish putative homologs from possible false positive hits. As a consequence, some BLAST hits are discarded that are in fact homologous.</p> <p>Results</p> <p>Analogous to the use of the genomics context in genome alignments, we test whether conserved functional context can be used to select candidate homologs from insignificant BLAST hits. We make a co-complex network alignment between complex subunits in yeast and human and find that proteins with an insignificant BLAST hit that are part of homologous complexes, are likely to be homologous themselves. Further analysis of the distant homologs we recovered using the co-complex network alignment, shows that a large majority of these distant homologs are in fact ancient paralogs.</p> <p>Conclusions</p> <p>Our results show that, even though evolution takes place at the sequence and genome level, co-complex networks can be used as circumstantial evidence to improve confidence in the homology of distantly related sequences.</p

Lysozyme Resistance in Streptococcus suis Is Highly Variable and Multifactorial

Background: Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen. Results: The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed th

Operon structure of Staphylococcus aureus

In bacteria, gene regulation is one of the fundamental characteristics of survival, colonization and pathogenesis. Operons play a key role in regulating expression of diverse genes involved in metabolism and virulence. However, operon structures in pathogenic bacteria have been determined only by in silico approaches that are dependent on factors such as intergenic distances and terminator/promoter sequences. Knowledge of operon structures is crucial to fully understand the pathophysiology of infections. Presently, transcriptome data obtained from growth curves in a defined medium were used to predict operons in Staphylococcus aureus. This unbiased approach and the use of five highly reproducible biological replicates resulted in 93.5% significantly regulated genes. These data, combined with Pearson’s correlation coefficients of the transcriptional profiles, enabled us to accurately compile 93% of the genome in operon structures. A total of 1640 genes of different functional classes were identified in operons. Interestingly, we found several operons containing virulence genes and showed synergistic effects for two complement convertase inhibitors transcribed in one operon. This is the first experimental approach to fully identify operon structures in S. aureus. It forms the basis for further in vitro regulation studies that will profoundly advance the understanding of bacterial pathophysiology in vivo

Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics

Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11β-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1

The association between medically unexplained physical symptoms and health care use over two years and the influence of depressive and anxiety disorders and personality traits: a longitudinal study

Background Medically unexplained physical symptoms (MUPS) are highly prevalent and are associated with frequent health care use (HCU). MUPS frequently co-occur with psychiatric disorders. With this study we examined the longitudinal association between MUPS and HCU over 2 years and the influence of depressive and anxiety disorders and personality traits on this association. Methods We analysed follow-up data from 2045 to 2981 participants from the Netherlands Study of Depression and Anxiety (NESDA), a multisite cohort study. The study population included participants with a current depressive and/or anxiety disorder, participants with a lifetime risk and/or subthreshold symptoms for depressive and/or anxiety disorders and healthy controls. HCU, measured with the Trimbos and iMTA questionnaire on Costs associated with Psychiatric illness (TIC-P), was operationalized as the number of used medical services and the number of associated contacts. MUPS were measured with the Four Dimensional Symptoms Questionnaire, depressive and anxiety disorders with the Composite International Diagnostic Interview and personality traits with the NEO Five-Factory Inventory. Measurements were taken at baseline, 1 and 2 years follow-up. We used generalized estimating equations (GEE), using HCU at all three measurements as (multivariate) outcome. GEE also takes into account the dependency of observations within participants. Results MUPS were positively associated with HCU over 2 years (medical services: RR 1.020, 95 % CI 1.017–1.022; contacts: RR 1.037, 95 % CI 1.030–1.044). Neuroticism and depression had the strongest influence on the associations. After adjustment for these factors, the associations between MUPS and HCU weakened, but remained significant (services: RR 1.011, 95 % CI 1.008–1.014; contacts: RR 1.023, 95 % CI 1.015–1.032). Conclusions Our results show that MUPS were positively associated with HCU over 2 years, even after adjusting for depressive and anxiety disorders and personality traits

Pulmonary artery remodeling differs in hypoxia- and monocrotaline-induced pulmonary hypertension

In the present study we analyzed structural characteristics of muscular pulmonary arteries and arterioles in two classic models of pulmonary hypertension, the rat hypoxia and monocrotaline models. We hypothesized that an increase in medial cross-sectional area would result in reduction of the lumen area and that these parameters would correlate with the increase in pulmonary artery pressure (PAP). Four weeks after a single injection of monocrotaline (MCT) or after 4 wk of hypoxic exposure the rats were killed. Both MCT and chronic hypoxia induced right ventricular hypertrophy. In separate groups of rats both MCT and chronic hypoxia increased PAP. MCT increased the media cross-sectional area of pulmonary arteries with an external diameter between 30–100 �m and 101–200 �m and reduced the lumen area of pulmonary arteries with an external diameter between 101–200 �m. Chronic hypoxia only slightly increased the media cross-sectional area without a change of the lumen area. Both MCT and hypoxia increased the percentage of partly muscularized and muscularized arterioles. The angiotensin-converting enzyme (ACE) inhibitor captopril (0.5 mg/kg/h) had no effect on MCT-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary artery remodeling. In chronic hypoxic rats it prevented an increase in medial cross-sectional area of pulmonary arteries with an external diameter between 30–100 �m and attenuated the increase in the percentag