Nutrient Limitation Governs Staphylococcus aureus Metabolism and Niche Adaptation in the Human Nose

Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.     

Overweight/Obesity and Respiratory and Allergic Disease in Children: International Study of Asthma and Allergies in Childhood (ISAAC) Phase Two

BackgroundChildhood obesity and asthma are increasing worldwide. A possible link between the two conditions has been postulated.MethodsCross-sectional studies of stratified random samples of 8–12-year-old children (n = 10 652) (16 centres in affluent and 8 centres in non-affluent countries) used the standardized methodology of ISAAC Phase Two. Respiratory and allergic symptoms were ascertained by parental questionnaires. Tests for allergic disease were performed. Height and weight were measured, and overweight and obesity were defined according to international definitions. Prevalence rates and prevalence odds ratios were calculated.ResultsOverweight (odds ratio = 1.14, 95%-confidence interval: 0.98; 1.33) and obesity (odds ratio = 1.67, 95%-confidence interval: 1.25; 2.21) were related to wheeze. The relationship was stronger in affluent than in non-affluent centres. Similar results were found for cough and phlegm, rhinitis and eczema but the associations were mostly driven by children with wheeze. There was a clear association of overweight and obesity with airways obstruction (change in FEV1/FVC, −0.90, 95%-confidence interval: −1.33%; −0.47%, for overweight and −2.46%, 95%-confidence interval: −3.84%; −1.07%, for obesity) whereas the results for the other objective markers, including atopy, were null.ConclusionsOur data from a large international child population confirm that there is a strong relation of body mass index with wheeze especially in affluent countries. Moreover, body mass index is associated with an objective marker of airways obstruction (FEV1/FVC) but no other objective markers of respiratory and allergic disorders.     

Matrix Conditions and KLF2-Dependent Induction of Heme Oxygenase-1 Modulate Inhibition of HCV Replication by Fluvastatin

Background & Aims

HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.

Methods

HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.

Results

All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.

Conclusions

Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.     

Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates

High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2) are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106). Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.     

Spermatozopsis acidophila Kalina (Chlorophyta,Volvocales), a little known alga from highly acidic environments

Abstract

A strain of the alga Spermatozopsis acidophila from highly acidic environments in the solfatara at Pisciarelli (Naples, Italy) has been studied from the ecological, physiological, morphological and ultrastructural points of view.

Its pH optimum (1.0) and its upper limit of pH (1.8) are the lowest recorded for any algal species.

Our ultrastructural investigation has confirmed that it belongs to the family Poly-blepharidaceae (Volvocales). A conspicuously developed chloroplast was noted, along with a remarkable quantity of microtubules and E. R. cisternae.     

Determination of isocyanate biomarkers in construction site workers

4,4′-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate in the manufacture of polyurethanes, dyes, pigments and adhesives. High concentrations of isocyanates are a potent respiratory irritant. Therefore, it is important to develop methods to monitor exposure to such compounds. We monitored biological samples from 40 non-exposed and 45 exposed construction site workers. 4,4′-Methylenedianiline (MDA) and N-acetyl-4,4′-MDA (AcMDA) were determined from untreated urine (U-MDA, U-AcMDA) and MDA was analysed from acid-treated urine (U-MDA-tot). Haemoglobin (Hb) adducts of MDA (Hb-MDA) were determined in all workers. The levels of biomarkers decreased in the following order: U-MDA-tot>U-AcMDA>U-MDA>Hb-MDA. The same order was found for the percentage of samples, which were found positive in exposed workers: 100%, 91%, 91%, 27%. The urine levels U-MDA-tot correlate with U-MDA, U-AcMDA and Hb-MDA with r=0.79, 0.86 and 0.39, respectively (Spearman rank order, p<0.01). U-AcMDA correlates with U-MDA and Hb-MDA with r=0.77 and 0.47, respectively (p<0.01). U-MDA correlates with Hb-MDA (r=0.38, p<0.05). The levels in the controls were significantly lower than in the exposed workers for all compounds (Mann–Whitney test, p<0.01). The median isocyanate-specific IgE-level was higher in the exposed workers, but the difference was statistically not significant. The change of the biomarker levels was compared in a group of workers (n=20), which were analysed prior to isocyanate exposure and after the exposure for ～4–7 months. All urine MDA metabolites and the Hb-adduct levels increased significantly (Wilcoxon sign test, p<0.01). Total IgE increased significantly after the exposure with isocyanate activity (p<0.01). With the present work it could be shown that outdoor workers are exposed to a similar extent as workers from a MDI factory.     

Radiosensitization of wildtype p53 cancer cells by the MDM2-inhibitor PXN727 is associated with altered heat shock protein 70 (Hsp70) levels

The oncoprotein MDM2 (murine double minute 2) is often overexpressed in human tumors and thereby attenuates the function of the tumor suppressor p53. In this study, we investigated the effects of the novel MDM2-inhibitor PXN727 on p53 activation, cell proliferation, cell cycle distribution and radiosensitivity. Since the localization of heat shock protein 70 (Hsp70) exerts different effects on radioresistance of tumor cells, we investigated the impact of PXN727 on intracellular, membrane, and secreted Hsp70 levels. We could show that PXN727 exerts its effects on wildtype p53 (HCT116 p53^+/+, A549) but not p53 depleted (HCT116 p53^−/−) or mutated (FaDu) tumor cells. PXN727 activates p53, induces the expression of p21, reduces the proportion of cells in the radioresistant S-phase and induces senescence. Radiosensitivity was significantly increased by PXN727 in HCT116 p53^+/+ tumor cells. Furthermore, PXN727 causes a downregulation of Hsp70 membrane expression and an upregulated secretion of Hsp70 in wildtype p53 tumor cells. Our data suggest that re-activation of p53 by MDM2-inhibition modulates Hsp70 membrane expression and secretion which might contribute to the radiosensitizing effect of the MDM2-inhibitor PXN727.     

Enhancement of Glucose Transport in Rat Thymocytes by Different Radical Sources

This study demonstrates that oxidative stress induced in rat thymocytes by the hydrophilic 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), the lipophilic cumene hydroperoxide (CumOOH) and the freely diffusible H₂O₂ is associated with an activation of facilitative glucose transport rate, mediated by GLUT1, the major transporter in this cell type. We compared the effects of the three tested radical sources on the kinetic transport parameters, showing that the transport rate enhancement in the treated cells can be ascribed to an increase in the V_max value, apart from the site of generation of the oxidative stress. The enhancement of glucose transport by the three oxidants in thymocytes was significantly attenuated both by protein tyrosine kinase inhibitors as genistein and tyrphostin A23 and by U73122, a phospholipase C inhibitor. Genistein and U73122 reversed also the cited increase of V_max values. It is concluded that the stimulation of glucose transport in response to different oxidants is mediated, at least in part, through reactive oxygen species (ROS)-induced stimulation of protein tyrosine kinase and phospholipase C pathways.     

TRPV1 Gates Tissue Access and Sustains Pathogenicity in Autoimmune Encephalitis

Multiple sclerosis (MS) is a chronic progressive, demyelinating condition whose therapeutic needs are unmet, and whose pathoetiology is elusive. We report that transient receptor potential vanilloid-1 (TRPV1) expressed in a major sensory neuron subset, controls severity and progression of experimental autoimmune encephalomyelitis (EAE) in mice and likely in primary progressive MS. TRPV1^−/− B6 congenics are protected from EAE. Increased survival reflects reduced central nervous systems (CNS) infiltration, despite indistinguishable T cell autoreactivity and pathogenicity in the periphery of TRPV1-sufficient and -deficient mice. The TRPV1⁺ neurovascular complex defining the blood-CNS barriers promoted invasion of pathogenic lymphocytes without the contribution of TRPV1-dependent neuropeptides such as substance P. In MS patients, we found a selective risk-association of the missense rs877610 TRPV1 single nucleotide polymorphism (SNP) in primary progressive disease. Our findings indicate that TRPV1 is a critical disease modifier in EAE, and we identify a predictor of severe disease course and a novel target for MS therapy.