Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis

Evaluation of Three Commercial PCR Assays for the Detection of Azole-Resistant Aspergillus fumigatus from Respiratory Samples of Immunocompromised Patients

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE®Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex®Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results

Evaluation of Three Commercial PCR Assays for the Detection of Azole-Resistant <i>Aspergillus fumigatus</i> from Respiratory Samples of Immunocompromised Patients

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE®Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex®Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results

Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes

Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp.▿ ‡

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment

World ocean review: Mit den Meeren leben 7. Lebensgarant Ozean – nachhaltig nutzen, wirksam schützen

Im Fokus der siebten Ausgabe des »World Ocean Review« stehen die Auswirkungen des Klimawandels auf die Physik des Meeres und auf seine Lebensgemeinschaften; die Folgen von Fischerei, Schifffahrt, Ressourcenabbau, Energiegewinnung und Meeresverschmutzung sowie die Fragen, wie sich Wirkstoffe aus dem Meer nutzen lassen und wie der Ozean künftig so verwaltet werden kann, dass sowohl sein Schutz als auch die Teilhabe möglichst aller Menschen an seinen Leistungen und Gütern gewährleistet sind

Longitudinal relationship between B-type natriuretic peptide and anxiety in coronary heart disease patients with depression

Objective: Patients with coronary heart disease (CHD) suffer from physical limitations, but also from psychological distress. Natriuretic peptides may be involved in the neurobiological processes that modulate psychological adaptation, as they are increased in heart disease and seem to have an anxiolytic-like function. Longitudinal data on this association are scarce. Methods: To assess the relationship between NT-proBNP and anxiety (Hospital Anxiety and Depression Scale (HADS)), we used secondary data from a multicenter trial from baseline to 24 months. Patients (N = 308, 80.8% male, mean age 60.1 years) had stable CHD and moderate levels of depression (HADS >= 8). Results: Multiple linear regression adjusted for age, sex, BMI, and physical functioning revealed NT-proBNP as a significant predictor for anxiety at baseline, 1, 6, 12, 18, and 24 months (all p < .05). Linear mixed model analysis with the six anxiety measures as level-1 variable and NT-proBNP as fixed factor revealed a significant time*NT-proBNP interaction (t(1535.99) = -2.669, p = .01) as well as a significant time*NT-proBNP*sex-interaction (1(1535.99) = 3.277, p = .001), when NT-proBNP was dichotomized into lowest vs. the three highest quartiles. Conclusion: Our results indicate a stable negative association of baseline NT-proBNP with anxiety over two years. In men and women, different pathways modulating this relationship appear to be in effect. Female patients with very low NT-proBNP levels, despite their cardiac disease, show persistently higher levels of anxiety compared to women with higher levels of NT-proBNP and compared to men