A “dock, lock, and latch” Structural Model for a Staphylococcal Adhesin Binding to Fibrinogen

AbstractGram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bβ chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic “dock, lock, and latch” mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria

There is more to accommodation of the eye than simply minimizing retinal blur.

Eyes of children and young adults change their optical power to focus nearby objects at the retina. But does accommodation function by trial and error to minimize blur and maximize contrast as is generally accepted? Three experiments in monocular and monochromatic vision were performed under two conditions while aberrations were being corrected. In the first condition, feedback was available to the eye from both optical vergence and optical blur. In the second, feedback was only available from target blur. Accommodation was less precise for the second condition, suggesting that it is more than a trial-and-error function. Optical vergence itself seems to be an important cue for accommodation

Prognostic Significance and Gene Expression Profiles of p53 Mutations in Microsatellite-Stable Stage III Colorectal Adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets

Probing Cellular Mechanoadaptation Using Cell-Substrate De-Adhesion Dynamics: Experiments and Model

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales

The Structure of Sortase B, a Cysteine Transpeptidase that Tethers Surface Protein to the Staphylococcus aureus Cell Wall

Many surface proteins of Gram-positive bacteria, which play important roles during the pathogenesis of human infections, are anchored to the cell wall envelope by a mechanism requiring sortases. Sortase B, a cysteine transpeptidase from Staphylococcus aureus, cleaves the C-terminal sorting signal of IsdC at the NPQTN motif and tethers the polypeptide to the pentaglycine cell wall cross-bridge. During catalysis, the active site cysteine of sortase and the cleaved substrate form an acyl intermediate, which is then resolved by the amino group of pentaglycine cross-bridges. We report here the crystal structures of SrtB_(ΔN30) in complex with two active site inhibitors, MTSET and E64, and with the cell wall substrate analog tripleglycine. These structures reveal, for the first time, the active site disposition and the unique Cys-Arg catalytic machinery of the cysteine transpeptidase, and they also provide useful information for the future design of anti-infective agents against sortases

Porous exfoliated poly(ε-caprolactone)/clay nanocomposites:Preparation, structure, and properties

Disclosed are the cna gene and cna-derived nucleic acid segments from Staphylococcus aureus, and DNA segments encoding cna from related bacteria. Also disclosed are Col binding protein (CBP) compositions and methods of use. The CBP protein and antigenic epitopes derived therefrom are contemplated for use in the treatment of pathological infections, and in particular, for use in the prevention of bacterial adhesion to Col. DNA segments encoding these proteins and anti-(Col binding protein) antibodies will also be of use in various screening, diagnostic and therapeutic applications including active and passive immunization and methods for the prevention of bacterial colonization in an animal such as a human. These DNA segments and the peptides derived therefrom are contemplated for use in the preparation of vaccines and, also, for use as carrier proteins in vaccine formulations, and in the formulation of compositions for use in the prevention of S. aureus infection.U

Collagen binding protein compositions and methods of use

Disclosed are the cna gene and cna-derived nucleic acid segments from Staphylococcus aureus, and DNA segments encoding cna from related bacteria. Also disclosed are Col binding protein (CBP) compositions and methods of use. The CBP protein and antigenic epitopes derived therefrom are contemplated for use in the treatment of pathological infections, and in particular, for use in the prevention of bacterial adhesion to Col. DNA segments encoding these proteins and anti-(Col binding protein) antibodies will also be of use in various screening, diagnostic and therapeutic applications including active and passive immunization and methods for the prevention of bacterial colonization in an animal such as a human. These DNA segments and the peptides derived therefrom are contemplated for use in the preparation of vaccines and, also, for use as carrier proteins in vaccine formulations, and in the formulation of compositions for use in the prevention of S. aureus infection.U