Isolation and characterization of a putative collagen receptor from Staphylococcus aureus strain Cowan 1.

In a previous study we demonstrated that cells of Staphylococcus aureus strain Cowan bind 125I-collagen in a receptor-ligand type of interaction (Speziale, P., Raucci, G., Visai, L., Switalski, L.M., Timpl, R., and Hook, M. (1986) J. Bacteriol. 167, 77-81). In the present communication we report on the isolation and preliminary characterization of a putative collagen receptor from a lysate of S. aureus strain Cowan. Antibodies raised against a collagen receptor positive strain inhibit the binding of 125I-collagen to bacterial cells, whereas antibodies raised against a collagen receptor negative strain were without effect. Solubilized cell surface components did not exhibit any measurable affinity for collagen-Sepharose. However, the inhibitory effect of the antibodies against bacterial cells was neutralized by the lysate from a receptor-positive but not receptor-negative strain. A collagen receptor assay was designed based on this observation and used to develop a receptor purification protocol involving anion exchange chromatography, ammonium sulfate precipitation, and gel chromatography. Using this procedure a protein with an apparent Mr of 135,000 was purified. This protein which was present on a collagen receptor-positive strain but not on a receptor-negative strain could completely neutralize the inhibitory activity of the antibodies raised against S. aureus strain Cowan. Furthermore, antibodies raised against the 135-kDa protein inhibited the binding of collagen to bacteria, and this protein is tentatively identified as a collagen receptor

Hatch Dates, Growth, Survival, and Overwinter Mortality of Age‐0 Alewives in Lake Michigan: Implications for Habitat‐Specific Recruitment Success

Alewives Alosa pseudoharengus are key components of Laurentian Great Lakes ecosystems and spawn in multiple habitat types. Exploration of alewife early life history dynamics within these different habitats should help identify important recruitment processes. During 2001‐2003, we quantified physical (temperature, transparency) and biotic (chlorophyll a, zooplankton densities) habitat factors and collected age‐0 alewives (using ichthyoplankton nets and trawls) in a nearshore region of Lake Michigan and Muskegon Lake, Michigan (a drowned river mouth lake connected to Lake Michigan). We characterized alewife hatch dates, individual condition, growth, mortality, and size‐dependent overwinter survival to infer differences in habitat‐specific recruitment success. Temperature, turbidity, chlorophyll‐a concentrations, and densities of zooplankton prey were consistently higher in Muskegon Lake than in nearshore Lake Michigan. On average, young alewives in Muskegon Lake hatched earlier, grew faster, were in better condition (based on a biphasic length‐weight relationship), and had greater survival than alewives in Lake Michigan. By the end of the growing season, young alewives in Muskegon Lake obtained a larger size than those residing in nearshore Lake Michigan, suggesting that they were more likely to survive through winter (a period of intense size‐selective mortality) and ultimately recruit to the adult population.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141621/1/tafs1298.pd

Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin

The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a \u27ladder\u27 pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen

Collagen-Mimetic Hydrogels Promote Human Endothelial Cell Adhesion, Migration and Phenotypic Maturation

This work evaluates the response of human aortic endothelial cells (HAECs) to thromboresistant collagen-mimetic hydrogel coatings toward improving the biocompatibility of existing off-the-shelf small-caliber vascular grafts. Specifically, bioactive hydrogels-previously shown to support α1/α2 integrin-mediated cell adhesion but to resist platelet activation-were fabricated by combining poly(ethylene glycol) (PEG) with a 120 kDa, triple-helical collagen-mimetic protein (Scl2-2) containing the GFPGER adhesion sequence. Analysis of HAECs seeded onto the resulting PEG-Scl2-2 hydrogels demonstrated that HAEC adhesion increased with increasing Scl2-2 concentration, while HAEC migration rate decreased over this same concentration range. In addition, evaluation of HAEC phenotype at confluence indicated significant differences in the gene expression of NOS3, thrombomodulin, and E-selectin on the PEG-Scl2-2 hydrogels relative to PEG-collagen controls. At the protein level, however, only NOS3 was significantly different between the PEG-Scl2-2 and PEG-collagen surfaces. Furthermore, PECAM-1 and VE-cadherin expression on PEG-Scl2-2 hydrogels versus PEG-collagen controls could not be distinguished at either the gene or protein level. Cumulatively, these data indicate the PEG-Scl2-2 hydrogels warrant further investigation as off-the-shelf graft coatings. In future studies, the Scl2-2 protein can potentially be modified to include additional extracellular matrix or cytokine binding sites to further improve endothelial cell responses

Assessing the energy implications of replacing car trips with bicycle trips in Sheffield, UK

A wide range of evidence supports policies which encourage people to cycle more and drive less, for health and environmental reasons. However, the likely energy implications of such a modal shift have remained relatively unexplored. In this paper we generate scenarios for increasing the cycling rate in Sheffield between 2010 and 2020. This is done through the novel application of a simple model, borrowed from population ecology. The analysis suggests that pro-cycling interventions result in energy savings through reduced consumption of fuel and cars, and energy costs through increased demand for food. The cumulative impact is a net reduction in primary energy consumption, the magnitude of which depends on a number of variables which are subject to uncertainty. Based on the evidence presented and analysed in this paper, we conclude that transport policy has a number of important energy implications, some of which remain unexplored. We therefore advocate the formation of closer links between energy policy and transport policy in academia and in practice; our approach provides a simple yet flexible framework for pursuing this aim in the context of modal shift