The metalloprotease SepA governs processing of accumulation‐associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare‐associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall‐anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA‐negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap‐dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT‐PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA‐related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap‐mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.     

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10¹ and 1.9 × 10⁴ cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.     

Intraspecific plant–soil feedback and intraspecific overyielding in Arabidopsis thaliana

Understanding the mechanisms of community coexistence and ecosystem functioning may help to counteract the current biodiversity loss and its potentially harmful consequences. In recent years, plant–soil feedback that can, for example, be caused by below‐ground microorganisms has been suggested to play a role in maintaining plant coexistence and to be a potential driver of the positive relationship between plant diversity and ecosystem functioning. Most of the studies addressing these topics have focused on the species level. However, in addition to interspecific interactions, intraspecific interactions might be important for the structure of natural communities. Here, we examine intraspecific coexistence and intraspecific diversity effects using 10 natural accessions of the model species Arabidopsis thaliana (L.) Heynh. We assessed morphological intraspecific diversity by measuring several above‐ and below‐ground traits. We performed a plant–soil feedback experiment that was based on these trait differences between the accessions in order to determine whether A. thaliana experiences feedback at intraspecific level as a result of trait differences. We also experimentally tested the diversity–productivity relationship at intraspecific level. We found strong differences in above‐ and below‐ground traits between the A. thaliana accessions. Overall, plant–soil feedback occurred at intraspecific level. However, accessions differed in the direction and strength of this feedback: Some accessions grew better on their own soils, some on soils from other accessions. Furthermore, we found positive diversity effects within A. thaliana: Accession mixtures produced a higher total above‐ground biomass than accession monocultures. Differences between accessions in their feedback response could not be explained by morphological traits. Therefore, we suggest that they might have been caused by accession‐specific accumulated soil communities, by root exudates, or by accession‐specific resource use based on genetic differences that are not expressed in morphological traits. Synthesis. Our results provide some of the first evidence for intraspecific plant–soil feedback and intraspecific overyielding. These findings may have wider implications for the maintenance of variation within species and the importance of this variation for ecosystem functioning. Our results highlight the need for an increased focus on intraspecific processes in plant diversity research to fully understand the mechanisms of coexistence and ecosystem functioning.     

α-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.     

Evaluation of detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> DNA in animal blood samples by quantitative PCR

Aim of the study: The purpose of this study was to find out the relationship between the phase of infection (acute or persistent) and the ability of quantitative PCR to detect DNA of Toxoplasma gondii in circulating leukocytes in blood. Methodology: Animal serum samples were examined (50 sheep, 47 dogs, 32 dairy cows, 91 wild boars and 36 rabbits) for the occurrence of IgM and IgG antibodies to T. gondii by ELISA. Uncoagulated blood samples from the same animals were examined for the detection of T. gondii DNA in circulating leukocytes by real-time PCR. Results: Only IgM antibodies, characteristic for acute infection, were detected in 45 of the 256 serum samples (17.6%). Only IgG antibodies, corresponding with chronic infection, were detected in 120 of the 256 samples (46.8%). In 91 of the 256 samples (35.5%) neither IgM or IgG were detected by ELISA. For real-time PCR, animals were divided into three groups based on the serological results: (group I — acute infection, group II — chronic infection, and group III — no infection). In group I, the presence of T. gondii DNA was detected in 9 out of 45 samples (20%), whereas in group II only 1 of 120 samples was positive for T. gondii DNA (0.8%). In group III, no DNA of T. gondii (0/91 samples) was detected by real-time PCR. Significance: The proof of DNA by real-time PCR in IgM positive samples was statistically significant in comparison to IgG positive samples (P<0.0001).     

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient‐sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB‐4, HL‐60 and KG‐1) and their impact on autophagy and survival was characterized. Data show that whereas KG‐1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co‐activation of AMPK and mTORC1 associated with increased autophagy, NB‐4 and HL‐60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG‐1 cells’ survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti‐leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.     

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.     

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.