Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs'' aggregation into amyloid fibers modulates their biological activity and role in biofilms.     

Lysis of Staphylococcus aureus Cell Walls by a Soluble Staphylococcal Enzyme

Enzyme preparations of Staphylococcus aureus were examined for their ability to solubilize (32)P-labeled cell walls of the parent organism. Enzymatic activity was observed in the growth medium, in soluble fractions, and associated with native cell walls. Enzyme associated with isolated cell walls could be inactivated with formaldehyde without reducing the susceptibility of the walls to the action of added enzyme. When cells are frozen and thawed, 50 to 75% of the intracellular enzyme is released along with 2% of the intracellular protein. This freeze-thaw extracted enzyme has little, if any, activity on intact S. aureus cells. It appears that the enzyme resides near the cell wall and acts on the cell-wall inner surface.     

Thermal Inactivation of Staphylococcal Enterotoxins B and C

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.     

Thermal Inactivation of Staphylococcal Enterotoxin B in Veronal Buffer

The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 mug/ml of this toxin was reduced to less than 0.7 mug/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole.     

Buoyant Density Analysis of Staphylococcal Bacteriophage 80 Transducing Particles

Densities of pase, tet, and ol transducing particles were indistinguishable, and about 0.002 g x cm(-3) less dense than the plaque-forming units (PFU). The densities of the PFU and nov transducing particles were indistinguishable, the PFU having a density of 1.507 g x cm(-3).     

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19–20 (FH19–20) and 5–7 (FH5–7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5–7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5–7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5–7. We found that although FH5–7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5–7 domains. Furthermore, binding of FH5–7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5–7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5–7 and regulates alternative pathway activation on plasma HDL particles.     

Viral Inactivation of Intramuscular Immune Serum Globulins

A new process for the production of intramuscular immunoglobulin products is described which includes viral inactivation through solvent-detergent treatment. Removal of solvent-detergent was accomplished by precipitation, filtration and diafiltration. Process-scale preparations had appropriate antibody potency levels, and improved IgG integrity relative to traditional IGIM products. Moreover, acceptable results were obtained in all in vitro and in vivo pre-clinical toxicology testing, as well as clinical evaluation. Scaled-down experiments demonstrated that the new process provides effective viral inactivation. Taken together, these results indicate that the new products should have the same efficacy of the previous IGIM products albeit with safety and processing improvements.     

Inactivation of the Bactericidal Activity of Human Serum by Liquoid (Sodium Polyanetholsulfonate

Four serum-sensitive strains of Escherichia coli were exposed to 10, 20, and 50% fresh, heat-inactivated, and fresh human serum to which had been added Liquoid at a final concentration of 0.05, 0.025, 0.0125 and 0.006%. It was found that 50% fresh serum (in nutrient, Mueller-Hinton, thioglycolate, or Trypticase Soy Broth) killed more than 10(4) organisms/ml within 3 min, whereas 20 and 10% fresh serum required up to 20 and 40 min, respectively, to kill a comparable number of organisms. To neutralize the activity of 50% fresh serum, 0.0125% Liquoid had to be added, whereas an 0.006% final concentration of Liquoid was sufficient to antagonize the activity of 10 and 20% serum. However, when exposing extremely small bacterial inocula to fresh serum, at least 0.025% Liquoid was necessary to abolish the serum-bactericidal activity of 20 and 50% fresh serum. Liquoid had to be added to 50% fresh serum within seconds to prevent the killing of the majority of test organisms derived from small inocula. It is recommended that blood samples drawn from septicemic or bacteremic patients be aseptically added to a suitable broth which contains at least 0.025% Liquoid in order to improve the chances of isolating pathogens present in small numbers.     

金黄色葡萄球菌肠毒素 C2 的基因克隆、 表达及其生物学活性

利用 PCR 技术从金黄色葡萄球菌的基因组 DNA 中克隆 SEC2 全长基因, PCR 产物与 pGEM-T 载体连接,经测序证实后进行亚克隆,构建其表达载体 pET-28a-SEC2 ,在大肠杆菌 BL21(DE3) 中表达成熟重组蛋白 (rSEC2) , 纯化 rSEC2 蛋白并对其生物学活性进行研究 . 结果表明：成功克隆了 SEC2 全长基因,测序证实该基因共 717 bp ,编码 239 个氨基酸,与 GenBank 中收录的 SEC2 成熟蛋白质序列完全一致, SEC2 基因登录 GenBank(Accession number ： AY450554) ; 构建了 SEC2 的表达载体 pET-28a-SEC2 ,并在大肠杆菌 BL21(DE3) 中得到高效可溶性表达,可溶性的 rSEC2 经 Ni²⁺ 亲和层析纯化达到电泳纯,纯化的 rSEC2 蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被 rSEC2 刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用 .     

Influence of pH on the Heat Inactivation of Staphylococcal Enterotoxin A as Determined by Monkey Feeding and Serological Assay

The effect of pH on the thermal inactivation of staphylococcal enterotoxin A was investigated. Analysis of heated toxin by immunodiffusion in gel indicated that enterotoxin A in beef bouillon was inactivated faster at pH 5.3 than at pH 6.2. The z values (slopes) for the heat inactivation curves at pH 6.2 and 5.3 were 49.5 and 55 F (about 27 and 30 C), respectively. Enterotoxin produced and heated in dialyzed Casamino Acids medium and assayed by monkey feeding was more easily inactivated by heat at pH 5.3 than at pH 7.8. Thermal inactivation curves for enterotoxin A in beef bouillon (5 mug/ml, pH 5.3) were determined by two methods, monkey feeding and serological assay. The z values for the curves obtained by these two methods were both 55 F, although loss of biological or toxic activity of the enterotoxin occurred before loss of serological activity.     

人可溶性低密度脂蛋白受体在甲醇酵母中的表达

为获得低密度脂蛋白受体配基结合结构域在甲醇酵母中的分泌表达 ,首先用RT PCR方法以人肝癌Bel 740 2总RNA为模板扩增了编码低密度脂蛋白受体配基结合结构域的基因片段。核酸测序分析表明克隆到的DNA片段的序列与报道的人LDLR的cDNA序列相同。然后构建了甲醇酵母表达质粒pPIC9K sLDLr ,并将其线性化后用电穿孔法导入PichiapastorisGS115。分别用SDS PAGE、Westernblot和Ligandbindingblot对GS115 pPIC9K sLDLr上清中的重组sLDLR进行鉴定。SDS PAGE和Westernblot分析表明表达的sLDLR的表观分子量为 36kD。Ligandbindingblot分析表明表达的sLDLR具有配基结合的生物学活性     

Effect of Meat and Isolated Meat Proteins on the Thermal Inactivation of Staphylococcal Enterotoxin B

The thermal inactivation of staphylococcal enterotoxin B was studied in a phosphate-saline buffer, in the presence of two meat proteins, myosin and metmyoglobin (MetMb), and in a ground-beef slurry. When enterotoxin B was incubated at temperatures from 60 to 110 C, it was shown that the initial thermal inactivation at 80 C was faster than at 100 or 110C. The heating of enterotoxin B at 60, 80, and 100 C in the presence of either myosin or MetMb resulted in a rapid loss of the enterotoxin. Thermal loss of the enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B in a ground round slurry was rapid when compared to inactivation in a phosphate-saline buffer. The rapid loss of enterotoxin B in the slurry may be due to a combination of thermal inactivation and the binding of enterotoxin molecules to meat proteins.     

Application of Physical Methods to the Study of Serum Lipoprotein

Abstract

One of the primary characteristics distinguishing prokaryotic from eukaryotic cells is the absence of a nucleus with a clearly defined nuclear membrane. In prokaryotic cells the DNA is condensed into a structure called the nucleoid. This structure has also been referred to attimes as the nuclear body, prokaryotic nucleus, bacterial chromosome, folded genome, or folded bacterial chromosome. The nomenclature sometimes becomes confusing because unfolded bacterial DNA free of other components of the nucleoid has also been referred to as the bacterial chromosome. To avoid such confusion, it would be preferable to reserve the terms nucleoid or bacterial chromosome to describe the condensed prokaryotic DNA structures which have some features analogous to the eukaryotic metaphase chromosome and condensed interphase chromatin. If this convention is followed, the terms “folded chromosome” or “folded genome” become ambiguous because they could equally mean “folded nucleoid.” These latter terms will, therefore, be avoided throughout this article.     

Chlorine Inactivation of Sphingomonas Cells Attached to Goethite Particles in Drinking Water

Bacteria in drinking water, attached or not attached to goethite particles, were disinfected with chlorine. No additional protection was provided to the bacteria by their attachment to particles, and the limited efficiency of inactivation by chlorine was attributed to the presence of bacterial aggregates in both types of suspension.     

金黄色葡萄球菌LukF-PV基因的原核表达及血清特异性抗体的检测

目的:在大肠杆菌中表达金黄色葡萄球菌(Staphylococcus aureus,SA)LukF-PV基因,纯化重组蛋白并以其为抗原检测儿童SA感染者血清特异性IgG抗体,分析不同部位SA感染患者血清LukF-PV抗体水平。方法:将Luk F-PV克隆至pET-28a(+)载体,IPTG诱导重组蛋白表达、His柱纯化重组蛋白后,用间接ELISA检测儿童SA感染者与健康对照者血清特异性IgG抗体水平。结果:成功表达纯化LukF-PV蛋白,儿童SA感染组与健康对照组血清特异性抗体均值分别为(0.309±0.063)、(0.505±0.261),具有统计学意义(P0.05)。不同部位来源标本的感染者中血液组和扁桃体组抗体OD均值分别为(0.634±0.225)、(0.481±0.264)与健康对照组有显著差异(P0.05),其余各组与健康对照组无显著差异(P0.05)。抗体阳性率统计分析中血液组分别与脑脊液组、扁桃体组、咽拭子组、痰液组有统计学意义(P0.05),其余各组间均无显著差异(P0.05)。结论:LukF-PV成功表达纯化,,儿童SA感染组LukF-PV特异性IgG抗体显著高于健康对照组,其中来源于血液和扁桃体部位的SA感染者抗体水平与健康者有显著差异,尤以血液感染者最显著。     

Differential Inactivation of Lymphocytes and Bone Marrow Stem Cells by Heterologous Anti-mouse γ-Globulin Serum

THE principal obstacle to the effective and widespread clinical application of allogeneic bone marrow transplantation, in the treatment of certain immunological deficiency diseases and leukaemia, is the occurrence of graft versus host (GVH) disease^1–3. This syndrome, resulting from donor cell-mediated immunological reactions against tissue antigens of the host, probably results from the presence of mature, immunologically reactive cells in the marrow inoculum, derived either adventitiously from, the circulating blood⁴ or as residents in the marrow. If these reactive cells could be separated out by physical means (compare ref. 5) or be inactivated specifically, still retaining the viability of the marrow stem cells, it should be possible to preclude at will the occurrence of the often fatal GVH disease, following allogeneic bone marrow transplantation.