Pseudomonas aeruginosa is an important pathogenic bacterium of nosocomial infections. The microbe easily produce biofilm which brings us much difficulties in clinical treatment. The formation processes of biofilm, including the stages of early bacteria planting, mushroom-like structure forming and extracellular matrix producing, are regulated by a series of molecules and genes. And quorum sensing system of the microbe is responsible for regulation of the whole process of biofilm formation. According to the process of biofilm formation and the mimitat associated regulation mechanism, several anti-biofilm therapeutic strategies have been applied in clinical medicine, and some novel drugs and methods are developed.
Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Polysaccharides, Bacterial ; metabolism ; Pseudomonas Infections ; drug therapy ; microbiology ; Pseudomonas aeruginosa ; genetics ; physiology ; Quorum Sensing ; genetics ; physiology

Xanthomonas campestris pv. campestris ( Xcc), causative agent of the black rot disease of cruciferous crops worldwide, produces large amount of extracellular polysaccharide( EPS), which has found wide applications in industry. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5gus A5, and a number of EPS-defective mutants were isolated. The Tn5gusA5 insertion sites in the mutants were analyzed by using thermal asymmetric interlaced PCR(TAIL-PCR), and the corresponding genes were identified by homology blast to the completely sequenced genome of Xcc 8004 strain. A novel gene, waxE, identified from the EPS-defective mutant 151D09, was found to be disrupted by the insertion of Tn5gusA5 in the open reading frame(ORF) with genome coordinates 4478998bp to 4479819bp.This gene showed 52% similarity to the kdtX gene of Serratia marcescens and 50% to the waaE of Klebsiella pneumoniae at amino acid level, with characteristics of glycostransferase 2 family domain. In order to identify the function of waxE gene, waxE gene deletion mutant of Xcc 8004 was constructed by gene replacement strategy in which waxE gene of genome was replaced by kanamycin resistant gene kan. The waxE gene deletion mutant strain, named Xcc 8570, was confirmed by both PCR and southern analysis. The growth rate of the deletion mutant 8570 in rich medium was not affected, but the EPS yield reduced by 35% as compared with the wildtype strain 8004. The deletion mutant could be completmented in trans with plasmid pLATC8976 harboring an intact waxE gene, and the EPS yield of the mutant was restored. The combined data showed that waxE gene involved in EPS biosynthesis in Xcc.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Blotting, Southern ; Cloning, Molecular ; DNA Transposable Elements ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polysaccharides, Bacterial ; genetics ; metabolism ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Xanthomonas campestris ; genetics ; metabolism

While ica gene of Staphylococcus epidermidis is known to undergo phase variation by insertion of IS256, the phenomenon in Staphylococcus aureus has not been evaluated. Six biofilm-positive strains were tested for the presence of biofilm-nega-tive phase-variant strains by Congo red agar test. For potential phase-variant strains, pulsed-field gel electrophoresis was done to exclude the possibility of contamination. To investigate the mechanism of the biofilm-negative phase variation, PCR for each ica genes were done. Changes of ica genes detected by PCR were confirmed by southern hybridization, and their nucleotides were analyzed by DNA sequencing. Influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane sur-face among a biofilm-negative phase-variant strain and its parent strain. A biofilm-negative phase-variant S. aureus strain was detected from 6 strains tested. icaC gene of the phase-variant strain was found to be inactivated by insertion of additional gene segment, IS256. The biofilm-negative phase-variant strain showed lower adher-ing capacity to polyurethane than its parent strain. This study shows that phase variation of ica gene occurs in S. aureus by insertion of IS256 also, and this biofilm-neg-ative phase variation reduces adhering capacity of the bacteria.
Bacterial Adhesion/*physiology ; Biofilms/*growth & development ; Cell Adhesion Molecules/genetics/*metabolism ; Comparative Study ; Equipment Contamination/prevention & control ; Mutagenesis, Insertional/methods ; Mutagenesis, Site-Directed/genetics ; Phase Transition ; Polysaccharides, Bacterial/genetics/*metabolism ; *Polyurethanes ; Species Specificity ; Staphylococcus aureus/cytology/*physiology ; Structure-Activity Relationship

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase-histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.
Bacteria ; enzymology ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Host-Pathogen Interactions ; Phosphorylation ; Polysaccharides, Bacterial ; biosynthesis ; Porphyromonas gingivalis ; enzymology ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Protein Tyrosine Phosphatases ; chemistry ; genetics ; metabolism ; Protein-Tyrosine Kinases ; chemistry ; genetics ; metabolism ; Quorum Sensing ; Signal Transduction ; Streptococcus gordonii ; enzymology ; Virulence Factors ; metabolism

Ebosin is a novel exopolysaccharide produced by Streptomyces sp.139 with remarkable activity against rheumatic arthritis in vivo. In this paper, we reported effects of Ebosin on the inflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in THP-1 cells. With the special fluorogenic peptide as substrates, the enzymatic activities of interleukin-1beta converting enzyme (ICE) and TNFalpha-converting enzyme (TACE) were inhibited by Ebosin separately. Using the real-time reverse transcription polymerase chain reaction (real-time PCR), the mRNA synthesis of the three cytokines were identified decline separately by Ebosin. The secretion quantum of three cytokines in THP-1 cells with Ebosin was lower than that of normal THP-1 cells determined by ELISA assay and Western blotting. All of these results showed that Ebosin has remarkably suppressed synthesis of the three cytokines in THP-1 cells through different pathways. The primary study of Ebosin on anti-inflammation mechanism was promoted developing the new drugs treating rheumatic arthritis.
ADAM Proteins ; metabolism ; ADAM17 Protein ; Anti-Inflammatory Agents ; pharmacology ; Caspase 1 ; metabolism ; Cell Line, Tumor ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Polysaccharides, Bacterial ; biosynthesis ; pharmacology ; RNA, Messenger ; metabolism ; Streptomyces ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo provide a basis for surface modification of polyelectrolyte multilayer films (PEM) on implants by exploring the effects of immobilization of PEM on titanium surfaces on their cell biological effects.

METHODSBy using plasmid of bone morphogenetic protein-2 (pBMP-2)-loaded lipopolysaccharide-amine nanopolymersomes (pLNP) as cationic polyelectrolytes and hyaluronic acid (HA) as anionic polyelectrolytes. PEM were constructed on alkaline-heat treated titanium surfaces via layer by layer self-assembly(LbL) technique. A successive deposition of HA and pLNP on titanium surfaces was defined as one cycle of assembly. The morphology of titanium surface before and after assembly treatment was observed by scanning electron microscopy (SEM). The ultraviolet (UV) spectra and surface contact angles of PEM with different self-assembly cycles were measured. The adhesion and proliferation of mesenchymal stem cell (BMSC) on surfaces of group A (4 cycles of assembly, with outermost layer of pLNP), group B (4.5 cycles of assembly, with outermost layer of HA), blank control (polished titanium, Ti group) and alkaline-heat treated titanium (Ti-OH group) were investigated. Cell differentiation indexed by alkaline phosphatase activity (ALP) and in situ transfection of BMSC on surfaces of group A, Ti, negative control [4 cycles of assembly without pBMP- 2] were evaluated.

RESULTSSelf assembly of PEM made the titanium surface become relatively smooth. DNA absorption peaked appear at 260 nm in UV spectra, and the absorption intensity increased with assembly, suggesting the successful construction of PEM. After alkali- heat treatment, the surface contact angle of titanium decreased from (62.6±4.9) ° to (8.1±2.2) °. During LbL, with alternately introducing pLNP and HA, the contact angle increased in a jagged mode at a gradually decreased rate, which further proved the success of assembly. Cell adhesion for group A at 0.5 and 1 h was 0.415±0.085 and 0.426±0.048, which was significantly higher than those for group B (0.299±0.012, 0.355±0.022), Ti-OH group (0.225±0.007, 0.260±0.010) and Ti group (0.302±0.056, 0.339±0.028) (P < 0.01). Cell proliferation for group A and B at 3, 5 and 7 d were significantly higher than those for Ti and Ti-OH group (P < 0.01). ALP in group A at day 7 was 261±58, which was significantly higher than those in group B and Ti group. Group A had in situ transfection capability for BMSC.

CONCLUSIONSPEM with good cell biological effects can be constructed successfully on titanium surfaces using gene- loaded lipopolysaccharide- amine nanopolymersomes and hyaluronic acid.

Alkaline Phosphatase ; metabolism ; Bone Morphogenetic Protein 2 ; genetics ; Cell Adhesion ; Cell Differentiation ; Cell Proliferation ; DNA ; Hyaluronic Acid ; chemistry ; pharmacology ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Microscopy, Electron, Scanning ; Nanoparticles ; chemistry ; Plasmids ; Polysaccharides, Bacterial ; pharmacology ; Prostheses and Implants ; Surface Properties ; drug effects ; Titanium ; chemistry ; Transfection