Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro . By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis , we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.     

Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU_αβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140^o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk_i) even though their binding site sizes differ greatly; the most probable values of Sk_i on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Sk_i values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk_i values are proposed.     

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

Overexpression of torsinA in PC12 cells protects against toxicity

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

Hierarchical Phosphorylation of Recombinant Tau by the Paired-Helical Filament-Associated Protein Kinase Is Dependent on Cyclic AMP-Dependent Protein Kinase

Abstract : Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl₂ and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr³⁶¹ and Ser⁴¹² in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser³⁵⁶ and Ser⁴⁰⁹ in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.     

DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

The Prophage of SPβc2DcitK1, a Defective Specialized Transducing Phage of BACILLUS SUBTILIS

The defective specialized transducing phage SP beta c2dcitK1 carries two known bacterial genes, kauA and citK, as well as SP beta hage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SP beta c2dcitK1 transduces SP beta-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSP beta-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and of the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SP beta c2 or by the clear-plaque mutant SP beta c1, the resulting double lysogen can be heat induced to release high-frequency-of-transduction (HFT) lysates for kauA and citK.     

Identification of proteins capable of metal reduction from the proteome of the Gram‐positive bacterium Desulfotomaculum reducens MI‐1 using an NADH‐based activity assay

Understanding of microbial metal reduction is based almost solely on studies of Gram‐negative organisms. In this study, we focus on Desulfotomaculum reducens MI‐1, a Gram‐positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. Using non‐denaturing separations and mass spectrometry identification, in combination with a colorimetric screen for chelated Fe(III)‐NTA reduction with NADH as electron donor, we have identified proteins from the D. reducens proteome not previously characterized as iron reductases. Their function was confirmed by heterologous expression in Escherichia coli. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins identified are NADH : flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase flavin adenine dinucleotide/NAD(P)‐binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble protein fraction, suggesting a type of membrane association, although PSORTb predicts both proteins are cytoplasmic. This study is the first functional proteomic analysis of D. reducens and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram‐positive bacterium.