Project description:Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal-contaminated mining sites. This tolerance is correlated with heritable increase of metal excretion efficiency; less pronounce cadmium induced growth reduction and, over-expression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and cadmium tolerant populations. Many cDNAs responded to cadmium exposure in a reference population. Significantly fewer clones were cadmium responsive in tolerant animals. Analysis of variance revealed transcripts that interact between cadmium exposure and population. Hierarchical clustering of these clones revealed two major groups. The first one contained cDNAs that were up regulated by cadmium in the reference culture, but non-responsive or down regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti-microbial defense, calcium-channel blocking, neurotransmitter transport, chromatin remodeling and, endoplasmatic vesicle activity. The second group consisted of cDNAs down regulated in reference animals but not responding or slightly up regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+ dependent stress signaling, proteolysis and digestion. The reference population showed a strong signature of stress-induced genome-wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon cadmium exposure. We confirmed the micro-evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress-adapted phenotype. Orchesella cincta (Collembola) from the laboratory population (LC) at the department of Animal Ecology, Vrije Universiteit Amsterdam were taken as the reference group. This population originated from a pine forest from the reference area (Roggebotzand, the Netherlands, latitude N = 52 º 34’17’’, longitude E = 5 º 47’56’’) that contains on average < 0.5 μg cadmium per gram litter and humus. Tolerant animals were collected from randomly selected litter samples at two areas of the abandoned, but still heavily polluted lead/zinc mining site of Plombières (Belgium, latitude N = 50˚44’03’’, longitude E = 5˚58’02’’): the locations contain an average cadmium concentration between 10 and 52 μg.g-1 soil (Lock et al. 2003; Sterenborg 2003; Van Straalen et al. 1987). To diminish putative environmental effects from the field the animals were reared in a climate room (20°C, 75% humidity, LD 12 h:12 h) in PVC jars on a moist plaster of Paris layer feeding on algae present on twigs for at least three generations before experimental treatment. Animals were exposed to cadmium (nominal concentration of 112,4 μg cadmium.g-1 dry algae) for 3 days, directly after molting, via algal food. Control groups received the same treatment except that water was spiked into the algal food instead of cadmium solution. The cadmium exposure concentration is two times the no observed effect concentration (NOEC) for reproduction in chronically exposed animals. Total RNA was extracted from times 12 pooled animals per treatment using SV Total RNA Isolation system (Promega) and quantified on a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). Total RNA was visualized on a 1.5% agarose gel to verify its integrity. Each experimental group (pool of 12 animals) was replicated 8 times. Dyes were swapped between biological replicates: 4 Cy-3 labeled replicates and 4 Cy-5 lebeled replicates per experimental group Microarray experiment was designed in a closed loop Lab culture clean vs Cd, Lab culture Cd vs Tolerant culture Cd, Tolerant culture Cd vs Tolerant culture clean, Tolerant culture clean vs lab culture clean

Project description:Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients receiving surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms it leads to refractory and persistent device-related infections (DRIs). Staphylococcal proportions within biofilms are more tolerant to antibiotics and immune responses, and thus are hard-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal and silicone surfaces. A bio-assay guided fractionation was performed to isolate the active compound (C3) from the crude SBT348 extract. Our results demonstrated that C3 effectively inhibits the growth (MIC: 31.25 µg/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 µg/ml) of S. epidermidis RP62A in vitro. Chemical characterization of C3 by heat and enzyme treatments, and High-Resolution Fourier Transform Mass Spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258. 3257 Da). Cytotoxicity profiling of C3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria melonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of C3 treated-S. epidermidis RP62A has further unmasked the negative effect of C3 on central metabolism (carbon, amino acid and protein, lipids, nucleotide and energy) suggesting its mode of action. Taken together, these findings suggest that C3 could be possibly used as antibacterial and antibiofilm coatings on medically-relevant surfaces and prevent the relapsing staphylococcal DRIs.

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Streptomyces sp. CdTB01

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