Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia

Streptococcus mutans, a major cariogenic bacterium, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. Mutant strains of S. mutans MT8148, defective in the major surface proteins glucosyltransferase (GTF) B-, C-, and D-, and protein antigen c (PAc), were constructed by insertional inactivation of each respective gene with an antibiotic resistant cassette. Susceptibility to phagocytosis was determined by analyses of interactions of the bacteria with human polymorphonuclear leukocytes, and the PAc-defective mutant strain (PD) showed the lowest rate of phagocytosis. Further, when PD and MT8148 were separately injected into the jugular veins of Sprague-Dawley rats, PD was recovered in significantly larger numbers and for a longer duration, and caused more severe systemic inflammation than MT8148, indicating that S. mutans PAc is associated with its systemic virulence in blood. Next, 100 S. mutans clinical isolates from 100 Japanese children and adolescents were analyzed by Western blotting using antisera raised against recombinant PAc, generated based on the pac sequence of MT8148. Four of the 100 strains showed no positive band and each exhibited a significantly lower phagocytosis rate than that of 25 randomly selected clinical strains (P < 0.01). In addition, three of the 100 strains possessed a lower molecular weight PAc and a significantly lower rate of phagocytosis than the 25 reference strains (P < 0.05). These results suggest that S. mutans PAc may be associated with phagocytosis susceptibility to human polymorphonuclear leukocytes, with approximately 7% of S. mutans clinical isolates possible high-risk strains for the development of bacteremia.     

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.     

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.     

Preparation of Staphylococcus aureus antigens for evaluation of their immunological reactivity with the human sera by the western blot method]

Extracellular antigens as well as cell wall extracts of 4 S. aureus strains isolated from different kinds of infection were analysed by Western-Blott technique. Materials obtained in two systems of bacteria cultivation (with and without aeration) were compared. Four systems of PAGE (native conditions, with 8.0 M urea, with SDS and SDS after previous reduction of the material with 2-mercaptoethanol) were compared in order to get the best differentiation of proteins and antigens. Immunological reactivity of the antigens mixture with two human sera: highly positive (with three S. aureus antigens in ELISA) from patient with staphylococcal sepsis and negative (from blood donor) were analysed. The best results were obtained after reduction of the cell wall extracted material in SDS-PAGE. The different protein patterns depending on the strain and the method of bacteria cultivation were observed. The standardisation of Western-Blott technique was performed, including titration of the sera to get the best differentiation of the antigens. The difference in immunological reactivity of the positive and negative sera with staphylococcal antigens mixture showed rather quantitative than qualitative character.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Changes in the photosynthetic apparatus of diatoms in response to low and high light intensities

The centric diatom Cyclotella cryptica and two strains of the pennate diatom Phaeodactylum tricornutum were grown under low and high light intensities (300 lux and 3,000 lux) over 4-6 weeks. Growth was monitored by repetitive cell count. The culture media were replaced weekly to avoid morphological and biochemical alterations caused by nutrient depletion. The ultrastructure of the cells was examined by transmission electron microscopy. Alterations in the light-harvesting antenna systems were investigated by Western immunoblotting. Both diatoms reduced the plastid area, i.e. decreased the amount of thylakoid lamellae, under high light intensity. The thylakoids still ran in groups of three with parallel orientation within the chloroplasts. The girdle band lamellae were not affected. The amounts of storage compounds and vacuoles increased. SDS-PAGE of total cell protein followed by Western immunoblotting with antisera directed against subunits of the light-harvesting antenna systems of C. cryptica (cc-antiserum) and the cryptophyte Cryptomonas maculata (cmac-antiserum) revealed that both diatoms reduced the amount of antenna polypeptides under increased light intensity. The cc-antiserum immunodecorated two bands with relative molecular masses (Mr) of 18,000 and 22,000 in C. cryptica. Both decreased under high light conditions to 67.2 +/- 6.1%. Five to seven bands in the Mr range of 14,000-27,000 were recognized in P. tricornutum. They decreased to 83 +/- 5.3%. Furthermore, the immunolabeling pattern for both strains differed under the two light regimes. The cmac-antiserum immunodecorated two polypeptides with Mr of 24,000 and 23,000 in C. cryptica, while both strains of P. tricornutum had five polypeptides in the Mr range of 14,000-24,000 that showed some differences in staining intensities between the two strains and in response to the light intensity applied.     

Electrophoretic and immunochemical characteristics of proteins of Rickettsia prowazekii strains of various virulence

PAAG-electrophoresis of the isogenic pair of Rickettsia prowazekii strains E and Evir lysates demonstrate the similarity in polypeptide tracks. The different electrophoretic mobility of the Mr 30 Kd protein from these strains as compared with the mobility of analogous protein from the standard virulent Breinl strain is registered. In immunoblot experiments the specific rabbit antiserums obtained on the 30th day of infection with the Breinl, E or Evir strains demonstrate the presence of the different main antigens 60 Kd or 70 Kd. The difference evidently reflects the specificity of development of two forms of infection by the strains having different virulence. The surface tris-soluble antigens of Rickettsia prowazekii have the similar polypeptide contents and immunochemical properties. The main component of tris-soluble antigens Mr 130 Kd protein is not strain specific having the common thermolabile epitope.     

Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

猪链球菌2型国内分离株毒力相关蛋白的分析

提纯猪链球菌 2型江苏分离株HA980 1的毒力相关蛋白溶菌酶释放蛋白 (muramini dase releasedprotein ,MRP)和胞外因子 (extracellularfacter,EF) ,制备其抗体供免疫转印之用。另提取猪源链球菌 1 7株国内分离株、1株德国分离株及 1株猪链球菌 2型人分离株的胞壁和胞外蛋白 ,经SDS PAGE ,与HA980 1株的MRP和EF的抗体作免疫转印。 1 1株MRP阳性 ,1 0株EF及EF 阳性 ,MRP及EF的分布存在 4种表型 :MPR+ EF+ (8 1 9) ,MRP+ EF (1 1 9) ,MRP+ EF- (1 1 9) ,MRP- EF- (1 0 1 9)。     

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.     

Involvement of the cell envelope of Listeria monocytogenes in the acquisition of nisin resistance

The involvement of the cell wall in the acquisition of nisin resistance by Listeria monocytogenes F6861 and its nisin-resistant mutant was investigated. Results indicated that without a cell wall, the acquired nisin resistance of the mutant was lost. Cell surface hydrophobicity was shown to correlate with nisin sensitivity; the wild type strain being more hydrophobic than its mutant. The possible role of S-layer proteins in nisin resistance was investigated. Examination of strains by freeze-etching and atomic force microscopy did not demonstrate the presence of S-layers in either strain while SDS-PAGE following S-layer extraction procedures revealed no major protein bands. Chloramphenicol did not adversely affect the frequency of isolation of nisin-resistant mutants, indicating that de novo protein synthesis was not involved. The involvement of other cell surface components, teichoic and lipoteichoic acids, was also examined. In contrast with other reports, comparison of the total phospholipid content of the mutant with its parental strain showed no significant difference ( P > 0.05).     

Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis, in addition to a number of antigens shared only by some strains.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Purification and characterization of the free form of the lactococcal extracellular proteinase and its autoproteolytic cleavage products

In addition to the cell wall proteinase, Lactococcus lactis subsp. cremoris produced significant amounts of a free extracellular proteinase. The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase. Both proteinase forms had a pH optimum between 4-6 and 5-8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related. Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50,000-80,000-fold increase in the specific activity of the free proteinase. It contained two major protein species (termed pro150 and pro115) with proteinase activity. As judged by SDS-PAGE, the Mr values of pro150 and pro115 were 150,000 and 115,000, respectively, and by chromatofocusing the isoelectric points were 4.3 and 4.1, respectively. Upon gel filtration, pro150 and pro115 had Mr values of 300,000 and 125,000, respectively, indicating that pro150 was a dimer and pro115 a monomer. Pro115 was an autodegradation product of pro150. Other distinct autodegradation products had Mr values of 90,000 (p90), 53,000 (p53), 37,000 (p37) and 30,000 (p30). These had little if any proteinase activity. Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase. Judging from its N-terminal sequence and Mr, p30 was derived from the C-terminal half of p53. Cleavage of pro150 to pro115 generated p37.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

Characterization of the Renibacterium salmoninarum haemagglutinin

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Antigenic Characterization of Chlamydia pneumoniae Isolated in Hiroshima,Japan

The morphology and antigenic property of elementary bodies (EBs) of new Chlamydia pneumoniae YK-41 strain isolated in Hiroshima, Japan, were compared with those of C. pneumoniae strains TW-183 and AR-39, C. trachomatis L2/434/Bu strain and C. psittaci Cal 10 and Budgerigar-1 strains by SDS-PAGE and immunoblotting techniques. In spite of a clear difference in EB morphology between the YK-41 and the other C. pneumoniae strains used, protein profile of the YK-41 strain in SDS-PAGE was similar to that of the other strains. However, some quantitative difference in 200 and 98 kDa peptides and a faint difference in SDS-PAGE pattern was also observed in the molecular masses from 42 to 50 kDa. Immunoblot analysis with the patient serum at the convalescent stage revealed the presence of genus-specific and species-specific antigens in YK-41 EBs: i. e., the major outer membrane protein and 73 kDa peptides were genus-specific and the peptides of 43, 46, 53, 60 and 98 kDa appeared to be C. pneumoniae-specific.     

Production of lysozyme-enriched biomass from cheese industry by-products

Cheese whey and cottage cheese whey are by-products of the milk and cheese industry, resulting from the production of cheese and cottage cheese (ricotta) from milk. They are still rich in organic substances and cannot be discarded into the environment without proper treatment. Whey and cottage cheese whey were used as culture media for some strains of the yeast Kluyveromyces lactis, transformed with the human lysozyme gene. It was found that the yeast strains grew well in both media and produced a considerable amount of recombinant protein. Production kinetics showed that the human lysozyme was produced in a greater amount within 36 h of fermentation (125 micrograms ml-1 vs 25 micrograms ml-1 in the control) than in the synthetic commercial media used for strain preparation and characterization. The recombinant protein produced was actually shown to be the human lysozyme, using renaturing SDS-PAGE and Western blot techniques. While producing recombinant protein, the Kluyveromyces strain cleared the cottage cheese whey of most organic substances and produced a considerable amount (almost 3%) of lysozyme-enriched useful biomass.