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Free and Hemophore-Bound Heme Acquisitions through the Outer Membrane Receptor HasR Have Different Requirements for the TonB-ExbB-ExbD Complex. Sylvie Létoffé, 2004.Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores . Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up . The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA . This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake . This requirement for more of the TonB complex is associated with a higher energy requirement . Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin . We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor . This dissociation is concomitant with heme uptake . We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport . Rifalazil Treats and Prevents Relapse of Clostridium difficile-Associated Diarrhea in Hamsters. Pauline M. Anton, 2004.Although vancomycin and metronidazole effectively treat Clostridium difficile-associated diarrhea and colitis (CDAD), their use is associated with a high incidence of relapsing C . difficile infection . Rifalazil is a new benzoxazinorifamycin that possesses activity against Mycobacterium tuberculosis and gram-positive bacteria . Here we compared rifalazil and vancomycin for effectiveness in preventing or treating clindamycin-induced cecitis in a hamster model of CDAD . Golden Syrian hamsters were injected subcutaneously with clindamycin phosphate (10 mg/kg), followed 24 h later by C . difficile gavage . Hamsters received by gavage for 5 days vehicle, vancomycin (50 mg/kg), or rifalazil (20 mg/kg) either simultaneously with (prophylactic protocol) or 24 h after C . difficile administration (treatment protocol) . While all vehicle-administered animals became moribund within 48 h of C . difficile administration, no rifalazil- or vancomycin-treated animals in either protocol showed signs of morbidity after 7 days . Ceca of rifalazil-treated animals showed absence of epithelial cell damage, significantly reduced congestion and edema, and less, but not statistically significantly less, neutrophil infiltration compared to those of vehicle-treated animals . In contrast, vancomycin-treated animals demonstrated severe epithelial cell damage and mildly reduced congestion and edema . Moreover, hamsters relapsed and tested C . difficile toxin positive (by enzyme-linked immunosorbent assay) 10 to 15 days after discontinuation of vancomycin treatment . None of the rifalazil-treated hamsters showed signs of disease or presence of toxins in their feces 30 days after discontinuation of treatment . Our results indicate that once daily rifalazil may be superior to vancomycin for curative treatment of CDAD . Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores. Maria Fluckinger, 2004.Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions . It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth . In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL) . Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities . In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores . By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions . Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections . Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays. Sung-Keun Rhee , 2004.To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance . This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences . Based on artificial probes, our results showed that under hybridization conditions of 50°C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity . Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes . The detection limit was  5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 107 cells in the presence of background RNA . Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r2 = 0.95 to 0.99) . Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions . While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms . In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation . The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r2 = 0.74) . In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures . Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity .
Degradation of a Caulobacter Soluble Cytoplasmic Chemoreceptor Is ClpX Dependent. Isabel Potocka, 2002.In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB . The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA . Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated . The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis . Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites . A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility . Structure of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A in Complex with Protein-Bound Methionine. Alexander B. Taylor, 2003. Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F. James J. Schmidt, 2003.The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known . High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities . Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths . Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F . In synthetic peptide substrates, the P1 and P3' residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively . By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher . Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient . The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a Ki value of 4 µM . In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs .
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