Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6539 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5-9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demonstration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four other solubilized membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.     

Randomness in crystallization of proteins from Staphylococcus aureus

Of many factors affecting protein crystallization, randomness in proteins has been given less attention although highly structured proteins would be at low entropy state. The factors, which impact on protein crystallization, are almost exclusively related to non-random amino acid properties such as physiochemical properties of amino acids. In this study, we used logistic regression and neural network to model the success rate of crystallization of 420 proteins from Staphylococcus aureus with each of non-random and random amino acid properties in order to determine whether randomness in a protein plays a role in the crystallization process. The results show that randomness is indeed involved in the crystallization process, and this rationale would enrich our knowledge on crystallization process and enhance our ability to crystallize more important proteins.     

金黄色葡萄球菌表面蛋白研究进展

金黄色葡萄球菌是一种常见的人兽共患病的病原菌。通过其表面蛋白(黏附素)与宿主的细胞外基质结合感染宿主,这些蛋白的结构已从分子水平上得到揭示。本综述了金葡菌产生的表面蛋白及其主要蛋白的分子结构。     

Antibacterial potential of Malaysian ethnomedicinal plants against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)

This study aimed to evaluate the antibacterial activities of 61 plant extracts from 49 Malaysian ethnomedicinal plants and to investigate the interaction of the active plant extracts in combination with synthetic antibiotics against the MSSA and MRSA strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the plant extracts were determined using a microdilution method against MSSA and MRSA strains. The interaction between active plant extracts and the antibiotics was assessed using the checkerboard method. The total fractional inhibitory concentration (∑FIC) indices from the combination were calculated to determine the nature of the interaction. Out of the 61 plant extracts tested against the MSSA strain, 7 plant extracts (̴ 11%) showed MIC values of less than 200 μg/mL, 17 extracts (̴ 28%) showed MIC between 200 and 800 µg/mL and seed extracts of Areca catechu showed MBC values of 400 μg/mL. The seed extract of A. catechu showed MIC and MBC of 400 μg/mL against the MRSA strains while leaf extract of Cocos nucifera showed MIC of 400 μg/mL against MRSA NCTC 12493. When the active plant extracts (MIC ≤ 200 µg/mL for MSSA, and ≤ 400 µg/mL for MRSA) were tested in combination with vancomycin and ciprofloxacin, they showed no interaction against both MSSA and MRSA with ∑FIC between 1.06 and 2.03. These findings provide a preliminary overview of the anti-MSSA and anti-MRSA properties of Malaysian ethnobotanical plants to combat Staphylococcal infections. Further research is needed to establish an antibacterial profile of the tested plant extracts.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .     

Isolation of five penicillin-binding proteins from Staphylococcus aureus

Abstract Five penicillin-binding proteins have been isolated from detergent-solubilized membranes of Staphylococcus aureus and have been separated from other membrane proteins by covalent affinity chromatography on 6-aminopenicillanic acid-Sepharose. The PBPs were resolved by electrophoresis on SDS-polyacrylamide gels and their ability to bind radioactive penicillin assayed after transfer from the polyacrylamide gel onto nitrocellulose membrane filters. Synthesis of a β-lactam antibiotic, propionylampicillin and of [2,3-³H]propionylampicillin of high specific activity greatly facilitated the assay of penicillin-binding activity.     

The polyphenol (-)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus

Aim: (?)‐Epicatechin gallate (ECg) modifies the morphology, cell wall architecture and β‐lactam antibiotic susceptibility of Staphylococcus aureus. As these effects result primarily from intercalation into the bacterial cytoplasmic membrane, the capacity of ECg to modulate the secretion of two key staphylococcal virulence factors, coagulase and α‐toxin, was examined. Methods and Results: Bioassays were used to determine coagulase and haemolysin activity in culture supernatants of a number of S. aureus isolates grown in the presence and absence of ECg; α‐toxin secretion was also evaluated by immunoblotting. Growth in ECg reduced the levels of activity of both proteins in culture supernatants; the effects could only be partly explained by ECg‐mediated inhibition of bioactivity and by induction of secreted proteases. Conclusion: ECg suppresses the secretion of coagulase and α‐toxin by clinical isolates of S. aureus. Significance and Impact of the Study: The observation that secretion of key components of staphylococcal virulence can be compromised by a naturally occurring polyphenol supports the notion that ECg and related compounds may have therapeutic utility for the control of infections that are currently difficult to treat due to the propensity of methicillin‐resistant S. aureus to accumulate antibiotic resistance genes.     

Different staphylococcal species contain various numbers of penicillin-binding proteins ranging from four (Staphylococcus aureus) to only one (Staphylococcus hyicus).

The penicillin-binding proteins of a total of 25 staphylococcal strains belonging to five different species were analyzed. All strains of the same species showed an identical penicillin-binding protein pattern which clearly differed from that of strains of the other species. Staphylococcus aureus, Staphylococcus intermedius, Staphylococcus simulans, and the dolphin strains were found to contain two to four penicillin-binding proteins. Strains of Staphylococcus hyicus exhibited only one penicillin-binding protein.     

Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus

The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of 2-DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.