Purification and characterization of diacetyl-reducing enzymes from Staphylococcus aureus.
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ABSTRACT:
SUBMITTER: Vidal I
PROVIDER: S-EPMC1149025 | biostudies-other | 1988 Apr
REPOSITORIES: biostudies-other
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Project description:Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins.We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays.BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity.BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules.BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.
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Project description:Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor in S. aureus and may be a therapeutic target for infectious diseases caused by S. aureus. For the purposes of anti-SAL drug development using structure-based drug design, X-ray crystallographic analysis of SAL overexpressed in Escherichia coli was performed. The recombinant protein was purified using a three-step protocol involving immobilized metal-affinity chromatography, cation-exchange chromatography and anion-exchange chromatography flowthrough methods, yielding 40?mg of protein per litre of bacterial culture. Crystals were obtained using the sitting-drop vapor-diffusion technique. Diffraction data to 3.0?Å resolution were collected on the BL44XU beamline at SPring-8 at the zinc peak of 1.2842?Å for SAD phasing. The crystals belonged to space group P4122 or P4322, with unit-cell parameters a = 131.0, b = 131.0, c = 250.6?Å, and are likely to contain four SAL molecules (408 residues) per asymmetric unit.
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Project description:On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5+/-0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6+/-0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000+/-5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a ;lysozyme' (endo-beta-N-acetylmuramidase) as previously suggested, but an endo-beta-N-acetylglucosaminidase.
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Project description:Staphylococcus aureus is a formidable pathogen capable of causing infections in different sites of the body in a variety of vertebrate animals, including humans and livestock. A major contribution to the success of S. aureus as a pathogen is the plethora of virulence factors that manipulate the host's innate and adaptive immune responses. Many of these immune modulating virulence factors are secreted toxins, cofactors for activating host zymogens, and exoenzymes. Secreted toxins such as pore-forming toxins and superantigens are highly inflammatory and can cause leukocyte cell death by cytolysis and clonal deletion, respectively. Coagulases and staphylokinases are cofactors that hijack the host's coagulation system. Exoenzymes, including nucleases and proteases, cleave and inactivate various immune defense and surveillance molecules, such as complement factors, antimicrobial peptides, and surface receptors that are important for leukocyte chemotaxis. Additionally, some of these secreted toxins and exoenzymes can cause disruption of endothelial and epithelial barriers through cell lysis and cleavage of junction proteins. A unique feature when examining the repertoire of S. aureus secreted virulence factors is the apparent functional redundancy exhibited by the majority of the toxins and exoenzymes. However, closer examination of each virulence factor revealed that each has unique properties that have important functional consequences. This chapter provides a brief overview of our current understanding of the major secreted virulence factors critical for S. aureus pathogenesis.
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Project description:As part of collaborative efforts to characterize virulence factors from Staphylococcus aureus, methods for the large-scale recombinant production of RNase HIII from S. aureus subspecies MRSA252 (Sa-RNase HIII) have been developed. RNase HIII-type ribonucleases are poorly characterized members of the RNase H group of endonucleases which hydrolyze RNA from RNA/DNA hybrids and are thought to be involved in DNA replication and repair. They are characterized by N-terminal extensions of unknown function that do not share sequence homology with the N-terminal extensions of bacterial RNases HI and RNases HII. Sa-RNase HIII was crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=48.9, b=74.2, c=127.5?Å, and diffracted to 2.6?Å resolution.
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Project description:BACKGROUND:The emergence of serious issues of multidrug resistance in the past few years have enforced the use of bacteriocins for combating infections. Threat posed to public health by various multidrug resistant (MDR) organisms can be resolved by discovering new antimicrobial proteins with broad spectrum of inhibition. RESULTS:In the current study, Bacteriocin (BAC-IB17) produced by Bacillus subtilis KIBGE-IB17 is found to be effective against different strains of methicillin resistant Staphylococcus aureus (MRSA). The approximate molecular mass of BAC-IB17 is 10.7?kDa. This unique bacteriocin is found to be highly thermostable and pH stable in nature. It also showed its stability against various heavy metals, organic solvents, surfactants and proteolytic enzymes. Amino acid profile of BAC-IB17 clearly showed that this protein mainly consists of non-polar and basic amino acids whereas; some acidic amino acids were also detected. Sequence of first 15 amino acid residues obtained from N-terminal sequencing of BAC-IB17 were NKPEALVDYTGVXNS. CONCLUSIONS:The anti-MRSA property of purified bacteriocin may be used to prevent the spread of MRSA infections. Remarkable features of BAC-IB17 suggests its applications in various pharmaceutical and food industries as it can function under a variety of harsh environmental conditions.
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Project description:Staphylococcus aureus is a pathogenic bacterium that causes a variety of mild to lethal human diseases. The rapid spread of multidrug-resistant strains makes the discovery of new antimicrobial agents critical. Dihydroorotase (PyrC), the third enzyme in the bacterial pyrimidine biosynthesis pathway, is structurally and mechanistically distinct from its mammalian counterpart. It has been confirmed to be essential in S. aureus making it an attractive antibacterial drug target. No protocol to express and purify S. aureus PyrC (SaPyrC) has been reported. To obtain the SaPyrC enzyme and overcome anticipated solubility problems, the SaPyrC gene was cloned into the pET-SUMO vector. The N-terminal His-SUMO fused SaPyrC was expressed in Escherichia coli BL21 (DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease. Purification of cleaved protein using HisTrap affinity and gel filtration columns resulted in native SaPyrC with estimated 95% purity and 40% yield. Both His-SUMO tagged and native SaPyrC form dimers, and enzyme characterization studies have shown that the His-SUMO tag affects enzyme activity slightly. Forward and reverse kinetic rate constants for both tagged and native SaPyrC were determined, and pH profiling studies revealed the optimal pH values for forward and reverse reactions.
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Project description:Nowadays antimicrobial lipopeptides are being widely exploited for developing potential therapeutic agents for treating bacterial infections. In the present study, we have purified and characterized an antimicrobial lipopeptide produced by Streptomyces amritsarensis sp. nov. (= MTCC 11845(T) = JCM 19660(T)). The lipopeptide was purified using silica gel chromatography, size exclusion chromatography and reverse phase- HPLC. The MS/MS analysis of the lipopeptide revealed that it has amino acid sequence as Ala-Thr-Gly-Ser-His-Gln and a long chain fatty acid tail with six times repeated the molecular mass of 161 Da which is corresponding to -C12H19. Based on the molecular mass (878.5 Da) and amino acid composition, the lipopeptide was identified as a novel lipopeptide. The MIC values of purified lipopeptide against Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), Mycobacterium smegmatis (MTCC 6) and clinical strain, Methicillin Resistant Staphylococcus aureus (MRSA) were found to be 10, 15, 25 and 45 μg/ml, respectively. It was completely stable at 70°C for 1 h and retained 81.8% activity after autoclaving (121°C for 15 min). It did not show any change in its activity profile between pH 5.0 - 9.0 and is stable to trypsin, proteinase K and lipase enzymes. It was found to be non-mutagenic against Salmonella typhimurium (TA98) and did not show cytotoxicity when checked against Chinese hamster ovary (CHO) cell line. In addition to antibacterial activity it also exhibits biosurfactant activity.
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Project description:BackgroundStaphylococcus aureus is a Gram-positive, pathogenic bacterium that causes a wide range of symptoms in humans and can form biofilm, which is a multicellular community of microorganisms that attaches to nonbiological and biological surfaces.MethodsHere, we aimed to isolate and characterize an S. aureus phage and examine the bactericidal activity alone and in conjunction with streptomycin treatment.ResultsWe isolated a virulent phage, WV, from a slaughterhouse in Jiangsu, China. This strain belonged to the family Myoviridae and presented a genome size of 141,342 bp. The optimal pH of the preservation buffer was 6-7, optimal growth temperature was 37°C, and optimal multiplicity of infection was 0.01. Phage WV can sterilize most clinical strains of S. aureus that had been isolated from clinical patients in the First People's Hospital of the Yunnan Province. Against low-concentration S. aureus culture, streptomycin demonstrated a greater antibiofilm effect than that of phage WV. By contrast, in high-concentration S. aureus culture, phage WV demonstrated greater antibiofilm effect than that of streptomycin. The use of phage WV and streptomycin together had a substantially greater overall antibiofilm effect than that achieved using either component alone.ConclusionThis study provides strong evidence for the effectiveness of phage application for the reduction of S. aureus biofilm growth and suggests that phages can be considered as a viable alternative to antibiotics in clinical settings.
| S-EPMC8619764 | biostudies-literature
Project description:BackgroundThe increased rate of resistance among two highly concerned pathogens i.e. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) necessitates the discovery of novel anti-MRSA and anti-VRE compounds. In microbial drug discovery, Streptomyces are well known source of two-thirds of natural antibiotics used clinically. Hence, screening of new strains of streptomycetes is the key step to get novel bioactive compounds with antimicrobial activity against drug resistant bacteria.ResultsIn the present study, Streptomyces antibioticus strain M7, possessing potent antibacterial activity against different pathogenic bacteria, was isolated from rhizospheric soil of Stevia rebudiana. 16S rRNA sequence of M7 (1418 bp) showed 96.47-100% similarity with different Streptomyces spp. and the maximum similarity (100%) was observed with Streptomyces antibioticus NBRC 12838T (AB184184). Phylogenetic analysis using neighbor joining method further validated its similarity with Streptomyces antibioticus NBRC 12838 T (AB184184) as it formed clade with the latter and showed high boot strap value (99%). Antibacterial metabolites isolated from the fermentation broth were characterized using NMR, FT-IR and LC-MS as actinomycins V, X2 and D. The purified actinomycins exhibited potent antibacterial activities against test bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, MRSA and VRE. Among these actinomycins, actinomycin X2 was more effective as compared to actinomycins D and V. The minimum inhibitory concentration values of purified compounds against a set of test bacterial organisms viz. VRE, MRSA, E. coli (S1-LF), K. pneumoniae sub sp. pneumoniae and B. subtilis ranged between 1.95 and 31.25 μg/ml.ConclusionsThis study demonstrates that actinomycins V, X2 and D produced by S. antibioticus strain M7 hold the potential to be used against multidrug resistant bacteria, particularly VRE and MRSA.
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