Phosphorylation of Glycerol in Staphylococcus aureus

Staphylococcus aureus dissimilates glycerol via an adenosine triphosphate-dependent kinase and not by the phosphoenolpyruvate phosphotransferase system.     

Widespread Arginine Phosphorylation in Staphylococcus aureus

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision–induced dissociation. Here we show that pArg is stable enough for commonly used Fe³⁺-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.     

Inactivation and Phosphorylation of Kanamycin by Drug-resistant Staphylococcus aureus

The cell-free system of clinical isolates of drug-resistant Staphylococcus aureus inactivated kanamycin, and the inactivated product was identified with kanamycin-3′-phosphate, in which the C-3-OH of the 6-amino-6-deoxy-d-glucose moiety of kanamycin was phosphorylated.     

Phosphorylation of a sugar-specific protein component of the lactose transport system in Staphylococcus aureus

A sugar-specific component of the lactose transport system in Staphylococcus aureus, Factor III^lac, is phosphorylated as an intermediate in the over-all transfer of a phosphoryl group from PEP to lactose. P-III^lac is isolated and shown to be a substrate for the final phosphoryl transfer reaction to sugar, catalyzed by Enzyme II^lac.     

耐甲氧西林金黄色葡萄球菌感染控制策略的新进展

耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus Aureus, MRSA)已成为一种越来越具有侵袭性和流行性的病原菌。通过染色体介导、质粒转移、基因表达调控和主动外排系统等途径,MRSA对包括万古霉素在内的多种抗生素产生了抗药性。从生态学和进化的角度来考虑,仅仅通过利用抗生素本身来解决耐药性的困境是不够的。这就迫使人们去寻找一类完全不同于化学药物治疗MRSA感染的机制。随着对免疫学和生物学认识的不断深入,基于免疫逃逸、细菌群体感应、基因调控等理论的发现,涌现了一批生物制剂抗MRSA感染的研究,相对于传统的抗生素治疗这是一个全新突破的领域。此外还有传统中草药的研发也提示其在抗MRSA方面存在积极的活力。本综述总结了生物制剂、新型策略化学药物和传统中草药治疗MRSA感染的最新进展,以寻找解决抗生素治疗困境的新线索。     

Two-Component Anti-Staphylococcus aureus Lantibiotic Activity Produced by Staphylococcus aureus C55

Staphylococcus aureus C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C₈ and C₁₈) chromatography. The yield following C₈ chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill S. aureus or Micrococcus luteus but not S. epidermidis strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.     

The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.The latest discoveries in the field of microbiology have proven that bacteria communicate between each other. In fact, many bacteria secrete small, diffusible signaling molecules. It is generally assumed that these molecules are used for a process termed “quorum sensing,“ the phenomenon whereby the accumulation of specific, diffusible, low-molecular-weight signal molecules (or “autoinducers”) enables bacteria to sense when the minimal number, or “quorum,” of bacteria for a concerted response to be initiated has been achieved (22). Gram-positive and Gram-negative bacteria use quorum-sensing communication circuits to regulate a diverse array of physiological activities, like symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation (22).The only presently known quorum-sensing mechanism that appears to be shared by both Gram-positive and Gram-negative bacteria is based on a group of interconvertible, diffusible molecules collectively referred to as autoinducer-2 (AI-2). The LuxS protein required for AI-2 production (19, 26) is a metal-containing enzyme (29) that cleaves S-ribosyl-l-homocysteine (SRH) to generate homocysteine and the AI-2 precursor, 4,5-dihydroxy-2,3-pentanedione (DPD). Outside the cell, unstable DPD undergoes chemical rearrangement that converts the molecule to AI-2, a small molecule able to penetrate membranes and to diffuse in the medium, allowing cross-species quorum sensing.The LuxS/AI-2 system in Staphylococcus aureus, a highly adaptable Gram-positive bacterium responsible for numerous clinical infections, has been analyzed in detail (13, 14, 28). Moreover, the emergence of antibiotic resistance has become a serious concern, especially due to methicillin-resistant S. aureus (MRSA) isolates that are resistant to all available penicillins and other β-lactam antimicrobial drugs (7). One appealing approach to this problem is to target bacterial systems associated with virulence mechanisms, particularly those based on signal transduction. Signal sensing leading to cellular responses must be tightly regulated to allow survival under variable conditions. The prevalent signaling mechanism in prokaryotes works through two-component systems. However, studies of the genomes of various pathogens have revealed a large family of eukaryotic-like Ser/Thr protein kinases (STPKs). It is becoming clear that signaling through Ser/Thr phosphorylation is a critical regulatory mechanism in pathogenic bacteria (16, 25). However, our understanding of S. aureus kinase biology has been seriously hampered by failure to identify relevant kinase substrates.While there is a significant body of published work defining the molecular mechanisms by which bacterial cells communicate, little is currently known about the regulation of the LuxS enzymes. The present study was undertaken to determine if the AI-2 producer protein LuxS might be regulated posttranslationally via STPK-dependent mechanisms.     

New Staphylococcus aureus genetic cluster associated with infectious osteomyelitis

Diverse genotyping methods, including multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), were used for genotyping Staphylococcus aureus in samples recovered from a clinical case of osteomyelitis. An unexpected genetic diversity of strains was determined, including four new sequence types (ST 1521, 1522, 1628 and 1629) belonging to the same genetic lineage, implying the appearance of a new subgroup derived from clonal complex CC121 isolated from that hospital. A close phylogenetic relationship among the STs was demonstrated, reflecting a possible diversifying evolution process. To our knowledge, there have no been previous reports of staphylococcal genetic variability observed within a single individual with such a high degree of variation. These findings emphasize the need for infection control measures to monitor the high genetic variability continuously occurring in this often dangerous infectious agent.     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.     

Evaluation of a New Bactericidal Spray Effective against Staphylococcus aureus

A new germicidal spray consisting of an active ingredient, 2-chloro-4-phenylphenol, solvent, and propellant was evaluated against aerosols of Staphylococcus aureus and spores of Bacillus subtilis. The method of evaluation consisted of determining the decay rate of the bacterial aerosols in a small chamber, with and without the aerial germicide present. The method is unique in that the effect of very short exposures of airborne bacteria to aerial germicides can be measured accurately. By use of the method outlined, extremely potent aerial germicides may be evaluated against highly sensitive organisms. The 2-chloro-4-phenylphenol spray was found to be extremely effective against S. aureus but only moderately effective against spores of B. subtilis.     

金黄色葡萄球菌肠毒素

金黄色葡萄球菌是一种重要的病原体,它产生多种类型的毒素,从而引起各种类型的疾病。金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins,SEs),是一组血清学上互不相同的热稳定肠毒素,有10个血清型。由于食入了被SEs污染的食品而主要引起肠胃炎,此外,SEs还是一种强的超抗原,它可以刺激非特异性T细胞增殖。SEs各型之间有着相似的结构和功能。     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.