bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane changes induced by exposure of Escherichia coli to human serum
Authors: Kroll H P , Bhakdi S , Taylor P W
Published: January 2020

The effect of bactericidal concentrations of lysozyme-free human serum on parameters of membrane integrity has been studied in serum-susceptible and serum-resistant Escherichia coli strains. Serum treatment released all of the alkaline phosphatase from the periplasmic space of two rapidly serum-susceptible strains but did so at different rates. In contrast, no periplasmic enzyme was released from two serum-resistant strains or from one moderately susceptible smooth strain. Lysozyme-free serum and heat-inactivated serum released comparable amounts of 86Rb+ from preloaded cells at comparable rates, regardless of serum susceptibility. Serum decreased the rate of phospholipid biosynthesis in both serum-susceptible and serum-resistant strains. In susceptible but not in resistant strains, intracellular ATP pools were depleted after serum exposure. Outer membranes and cytoplasmic membranes were prepared from serum-treated E. coli, and assays for C3 and C5b-9(m) were performed. With rapidly susceptible strains, C3 deposition on the outer membrane without attachment of C5b-9(m) occurred during the short prekilling phase. Subsequent bacterial killing was accompanied by deposition of C5b-9(m), which was recovered with C3 exclusively in outer membrane fractions with increased density and by eventual total loss of recoverable cytoplasmic membranes. Minimal deposition of complement components, without accompanying cytoplasmic membrane loss, occurred with serum-resistant strains. Loss of recoverable cytoplasmic membrane was not due to the action of either serum or bacterial phospholipase A. The results raise the possibilities that C5b-9(m) primarily damages the outer membrane and that the bacteria themselves actively participate in the ensuing, as yet unclarified, metabolic reactions that finally lead to their death.
Tracheal function during influenza infections
Authors: Nugent K M , Pesanti E L
Published: January 2020

Studies with animal models have demonstrated that viral respiratory tract infections suppress bacterial clearance processes in the lung and that this alteration in host defenses appears to explain the excessive mortality from bacterial pneumonia during influenza epidemics. However, since the pathogenesis of postinfluenza pneumonia and other pneumonias probably involves the aspiration of normal nasopharyngeal flora, injury to major airways associated with influenza infections could also contribute to the development of bacterial pneumonia by increasing bacterial deposition in the peripheral lung. We investigated this possibility by evaluating tracheal clearance processes and spontaneous changes in the tracheal flora in a murine model for acute influenza. During fine-particle aerosol exposures to Staphylococcus aureus, influenza-infected mice retained the same number of bacteria on their proximal tracheal surfaces as did control mice, and the relative adherence of the staphylococci to the trachea was similar in both groups of mice. However, the clearance of viable staphylococci from the trachea was significantly delayed in influenza-infected mice. Control and influenza-infected mice were also evaluated for changes in their normal tracheal flora. Mice with established influenza infections had more frequent spontaneous colonization with gram-negative bacteria, more bacterial isolates per animal, and higher bacterial concentrations in tracheal homogenates than control mice. These changes in tracheal flora were most pronounced on day 7 after virus inoculation and persisted after virus titers were undetectable, but eventually resolved by day 14 after virus infection. Tetracycline therapy started 2 days after virus inoculation prevented the increased colonization. This impaired clearance function and increased spontaneous colonization were associated with morphological evidence of mucosal regeneration. We conclude that spontaneous changes in tracheal flora occur during influenza infections, that these changes reflect, in part, impaired clearance functions, and that such changes could contribute to the development of pneumonia regardless of the clearance capacity of the lung parenchyma.
Toxofactor associated with Toxoplasma gondii infection is toxic and teratogenic to mice
Authors: Grimwood B , O'Connor G , Gaafar H A
Published: January 2020

A toxin associated with Toxoplasma gondii infection was obtained from the trophozoites and culture medium used to propagate the parasite in cell cultures. The toxin, named Toxofactor (TF), administered parenterally or nonparenterally in adult mice, produces transient symptoms of lethargy, ruffled fur, and body weight loss. Organ changes which accompanied the outward symptoms included hepatosplenomegaly and involuted thymus. TF activity was detected in extracts of the blood, peritoneal fluid, liver, and spleen of infected mice. Severe damage to embryonal and fetal development was induced when TF was administered during pregnancy. Resorption, abortion, and congenital abnormalities were produced, dependent upon the stage of development at the time of exposure. Adult mice which had reacted to and recovered from an initial intraperitoneal injection to TF were protected against a secondary challenge from TF. Fetal development was also protected from damage when TF was used to challenge adults previously exposed to TF. Mouse and rabbit anti-TF sera neutralized TF activity in the adult. In no instance did control mice show any deleterious effect when exposed to soluble cell lysate from the uninfected cell line (BHK-21) used to propagate the organism plus the used medium from these same uninfected cells. TF activity was not attributed to bacterial, myocoplasmal, or viral contamination. TF toxic activity is labile to elevated temperature and high or low pH, which also destroy its protective properties. TF activity was sensitive to trypsin and was obtained in the elution fraction (alpha-methyl-D-mannoside) from affinity chromatography (concanavalin A-Sepharose 4B). Ultrafiltration indicated the molecular weight to be between 50,000 and 100,000. TF, apparently a glycoprotein, was quantitated for activity by a weight loss assay. A unit of activity was defined as the minimum quantity of TF (highest dilution) which produced at least a 10% average body weight loss in adult Nya:NYLAR female mice between days 7 and 12 post-intraperitoneal injection.
Infection of mouse lymphoblastoid cell lines with Mycoplasma hyorhinis: complex nature of mycoplasma-host cell interactions
Authors: Butler G H , Stanbridge E J
Published: January 2020

Mycoplasma hyorhinis infection of lymphoid cells is a complex process. Mycoplasmas adsorb to cell surface receptors and undergo lateral redistribution on the cell membrane. This process culminates in the formation of co-caps of mycoplasmas and specific cell surface antigens. One or more of these antigens may be a M. hyorhinis receptor(s) or may bear a receptor moiety(s). We show that the cell surface antigens Thy-1.2 and Thy-1.1, and to a lesser extent H-2 and gp70, but not T200, are co-capped with M. hyorhinis on the membranes of acutely infected mouse thymic lymphoblastoid cell lines. These antigens may represent multiple receptor(s) for M. hyorhinis since there is no correlation between the expression of any individual antigen and the susceptibility of these cell lines to infection.
Exopolysaccharide production by viridans streptococci in experimental endocarditis
Authors: Mills J , Pulliam L , Dall L , Marzouk J , Wilson W , Costerton J W
Published: January 2020

Light and electron microscopy with histochemical staining were used to estimate exopolysaccharide production by strains of viridans streptococci recovered from patients with endocarditis. Six strains were selected for study because they represented a wide range of in vitro polysaccharide production. By light microscopy, there was good agreement between three polysaccharide stains (ruthenium red, periodic acid-Schiff and calcifluor white) in the amount of glycocalyx produced, which ranged from minimal (0 to 1+) to maximal amounts (4+). Two strains selected for minimal (strain 1) and maximal (strain 6) in vitro exopolysaccharide production were studied after we used them to experimentally infect cardiac vegetations. Glycocalyx could be demonstrated surrounding organisms in cardiac vegetations, and the relative amounts produced were similar to those seen in vitro. Vegetations formed by glycocalyx-producing strains were also larger than those formed by glycocalyx-deficient strains. Viridans group streptococci which produce exopolysaccharide in vitro also do so within cardiac vegetations. The relationship of exopolysaccharide production to maintenance of endocardial infection is discussed.
Specific Surface Antigens of SmT Variants of
Mycobacterium avium
Authors: Thorel Marie-Françoise , David Hugo L.
Published: January 2020

SmT variants of
Mycobacterium avium
ATCC 15769 formed novel antigens which were absent in SmD variants.
Molecular cloning of the temperature-inducible outer membrane protein 1 of Yersinia pseudotuberculosis
Authors: Bölin I , Wolf-Watz H
Published: January 2020

When Yersinia pseudotuberculosis is shifted from growth at 26 degrees C to growth at 37 degrees C, the synthesis of a plasmid-associated outer membrane protein, protein 1, is induced (Bölin et al., Infect. Immun. 37:506-512). The structural gene of this protein was found to be located on the virulence plasmid pIB1 of Y. pseudotuberculosis. One cosmid hybrid plasmid pBW8 was studied which carried a region of the virulence plasmid. This hybrid plasmid expressed in Escherichia coli K-12 a novel temperature-inducible outer membrane protein which is immunologically related to and has the same molecular weight as protein 1. Protein 1 was purified to homogeneity, and 14 amino acids of the N-terminal end were determined. From this sequence, the tentative corresponding DNA sequence was deduced, and a set of 11-nucleotide-long DNA probes was chemically synthesized. By using these probes in Southern blotting experiments, the genetic location of the N-terminal end of protein 1 was established. By introducing the transposon Tn5 into the virulence plasmid pIB1, mutants were obtained that did not express protein 1. One class of these mutants was still Ca2+ dependent and virulent, suggesting that protein 1 is not a major virulence determinant. Tn5-derived insertion mutants were also obtained which were Ca2+ independent. Such mutants were found to be avirulent. One Ca2+-independent mutant still expressed protein 1, indicating that the regulatory expression of protein 1 is not linked to Ca2+ dependence.
Specific inhibition of adsorption of Actinomyces viscosus T14V to saliva-treated hydroxyapatite by antibody against type 1 fimbriae
Authors: Clark W B , Wheeler T T , Cisar J O
Published: January 2020

Specific antibodies and their Fab fragments were used to study adsorption of Antinomyces viscosus T14V to saliva-treated hydroxyapatite. Antibody against A. viscosus T14V blocked adsorption of the strain to saliva-treated hydroxyapatite, and this activity was removed after preincubation of the immune immunoglobulin G (IgG) with purified type 1 fimbriae in amounts near equivalence but not after preincubation with type 2 fimbriae in antigen excess. Specific IgG or Fab fragments against type 1 fimbriae inhibited adsorption of strain T14V to saliva-treated hydroxyapatite and promoted desorption of cells already attached to the apatite surface. Anti-type 2 IgG or Fab fragments did not possess these activities. These data demonstrate that type 1 but not type 2 fimbriae mediate adsorption of strain T14V to the saliva-treated hydroxyapatite surface.
Teichoic acids of Streptococcus agalactiae: chemistry, cytotoxicity, and effect on bacterial adherence to human cells in tissue culture
Authors: Goldschmidt J C , Panos C
Published: January 2020

The ratio of teichoic acid to lipoteichoic acid (LTA) in a strain of Streptococcus agalactiae type III was found to be 8:1, with the total amount of LTA being 0.1% of the dry weight of the organism. Purified teichoic acid contained D-alanine and possibly a small amount of D-glucose and was approximately 22 glycerol phosphate units in length. The linkage between each of these units was 1-3. In addition, LTA contained a complex lipid, more glucose, and an unusually high content of a short-chain fatty acid, tridecanoic acid. This LTA was cytotoxic for a variety of human cell monolayers in tissue culture, including one derived from the human central nervous system. Established human cells were more sensitive than primary cell monolayers to this LTA, with as little as 12.5 micrograms of LTA per ml being cytotoxic for HeLa cells. Teichoic acid (250 micrograms/ml) was nontoxic under identical conditions. These cytotoxicity results suggest an LTA involvement in group B streptococcal pathogenesis. Also, the first model system for the study of group B streptococcal adherence to primary human embryonic amnion cells in tissue culture is detailed. This system was used to quantitate pronounced differences in tissue tropism between S. agalactiae and Streptococcus pyogenes and showed enhanced binding by this group A coccus over that of S. agalactiae for amnion cell monolayers. The adherence of both streptococcal species to only a portion (40%) of these amnion cells suggested that host cell receptor expression may vary for primary cells in vitro. Finally, this strain of S. agalactiae was shown to adhere to amnion cells by a non-LTA-mediated mechanism. The possibility of an LTA-mediated versus a protein-mediated adherence mechanism for host cells that is related to the virulence of S. agalactiae is discussed.
Escherichia coli O18ac antigen: structure of the O-specific polysaccharide moiety
Authors: Gupta D S , Jann B , Jann K
Published: January 2020

The O-specific polysaccharide moiety (O18ac polysaccharide) of the O18ac antigen (lipopolysaccharide) from Escherichia coli 2980 (O18ac:K5:Fim+:H-) was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G-50. The primary structure of the O18ac polysaccharide was elucidated by composition, fragmentation procedures, methylation analysis, and nuclear magnetic resonance spectroscopy. The polysaccharide consists of repeating units of the pentasaccharide: (formula; see text) which are joined in the polymer by alpha-1,2 linkages.
Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate
Authors: Shafer W M , Martin L E , Spitznagel J K
Published: January 2020

Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMNs) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, we pretreated the PMNs with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex and Sephadex G-75 chromatography resulted in the recovery of at least two antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition, and molecular weight (Mr, 37,000 and 57,000). As we have shown before (Shafer et al., Infect. Immun. 43:834-858), with unfractionated proteins, these two proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4' phosphate residue (Vaara et al., FEBS Lett. 129:145-149). The data suggest that at least two different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. Moreover, the availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules.
Effect of mouse lymphokines and cloned mouse interferon-gamma on the interaction of Rickettsia prowazekii with mouse macrophage-like RAW264.7 cells
Authors: Turco J , Winkler H H
Published: January 2020

We studied the effects of crude mouse lymphokines and cloned mouse interferon-gamma on the interaction of Rickettsia prowazekii with mouse macrophage-like RAW264.7 cells. Treatment of RAW264.7 cells with lymphokines before infection, after infection, or both before and after infection with R. prowazekii led to killing of a substantial proportion of the RAW264.7 cells. Such cytotoxicity required both lymphokines and viable R. prowazekii and did not occur in mouse fibroblastic L929 cells. Untreated cultures of RAW264.7 cells supported good growth of the Breinl strain of R. prowazekii, but in lymphokine-treated cultures, little or no rickettsial growth occurred in the cells that survived the cytotoxic reaction. In addition, treatment of RAW264.7 cells with lymphokines before rickettsial infection was associated with suppression of the initial infection. The effects of cloned mouse interferon-gamma were similar to the effects of crude mouse lymphokines. Assessment of cytotoxicity, inhibition of the initial infection, and inhibition of rickettsial growth in RAW264.7 cells pretreated with various concentrations of interferon-gamma indicated that the effects of the lymphokines could be explained by the interferon-gamma that was present in these preparations. Treatment of RAW264.7 cells with interferon-gamma makes them unsuitable host cells for R. prowazekii.
Augmentation of protective and antibacterial activity induced by muramyl dipeptides in CBA/N defective mice with X-linked immunodeficiency for Salmonella enteritidis infection
Authors: Onozuka K , Saito-Taki T , Nakano M
Published: January 2020

Stimulation of resistance induced by muramyl dipeptide (MDP) and its analog, N alpha-MDP-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)], was examined in experimental salmonellosis in CBA/N defective mice with X-linked immunodeficiency to virulent Salmonella enteritidis no. 11. An injection of either MDP or MDP-Lys(L18) did not induce any effective protection, but repeated injections of MDP-Lys(L18) (100 micrograms per mouse per day for 3 days consecutively) to the mice before bacterial challenge gave some protection. Multiple injections with MDPs once a day for several days consecutively strongly increased bactericidal capacity in the peritoneal cavities and spleens of the mice. Moreover, previous injection of the MDPs could elevate the phagocytic function of the reticuloendothelial system in the defective mice. These results indicate that nonspecific resistance of CBA/N defective mice to salmonella infection can be improved by previous administrations of MDPs.
Monoclonal antibody identifies circumsporozoite protein of Plasmodium malariae and detects a common epitope on Plasmodium brasilianum sporozoites
Authors: Cochrane A H , Collins W E , Nussenzweig R S
Published: January 2020

We produced a hybridoma secreting an immunoglobulin G1 monoclonal antibody against the circumsporozoite protein of the human malaria parasite Plasmodium malariae (Uganda 1/CDC). The monoclonal antibody produces a circumsporozoite precipitation reaction when incubated with viable sporozoites of P. malariae and reacts at high titers with heat-fixed sporozoites in an indirect immunofluorescent antibody test. Using the purified monoclonal antibody and Western blot analysis, we identified two polypeptides with apparent molecular weights of 60,000 (Pm 60) and 48,000 (Pm 48) in extracts of P. malariae sporozoites. Two-dimensional electrophoretic analysis of Pm 60 and the circumsporozoite protein Pm 48 indicated their isoelectric points to be acidic, with values of 5.3 and 4.7 to 5.0, respectively. A two-site immunoradiometric assay showed that the circumsporozoite protein recognized by the monoclonal antibody contains a repetitive epitope. P. malariae monoclonal antibody also reacted strongly with sporozoites of the simian parasite Plasmodium brasilianum, indicating a shared epitope on sporozoites of the two species. The P. malariae antibody did not bind sporozoite antigens of any other primate malarias, including Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale.
Interferon-gamma inhibits growth of Coxiella burnetii in mouse fibroblasts
Authors: Turco J , Thompson H A , Winkler H H
Published: January 2020

We studied the effects of various mouse interferon preparations on the growth of Coxiella burnetii in mouse fibroblasts. The addition of both recombinant interferon-gamma and a crude lymphokine preparation that contained interferon-gamma to infected L929 cells inhibited the growth of C. burnetii, whereas the addition of a crude preparation of type I interferons did not. Cycloheximide suppressed the inhibitory effects of recombinant interferon-gamma and crude lymphokines.
Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1
Authors: Valvano M A , Crosa J H
Published: January 2020

The aerobactin-mediated iron uptake system encoded by pColV-K30 and other ColV plasmids has been associated with the ability of Escherichia coli strains to cause disease. We investigated whether the pColV-K30 aerobactin system is present in E. coli K1 VW187 isolated from a human neonate with meningitis. This strain exhibited a functional aerobactin-mediated iron uptake system, as assessed by a cross-feeding bioassay and by its sensitivity to cloacin, a bacteriocin that recognizes the outer membrane receptor for iron-aerobactin complexes. By using a variety of techniques, we could not find any plasmid harboring the aerobactin genes. Hybridization of restriction endonuclease-cleaved chromosomal DNA from strain VW187 with various clones containing subsets of the pColV-K30 aerobactin region showed that the aerobactin genes were located on a 10.5-kilobase-pair chromosomal HindIII restriction fragment which also contained IS1-like insertion sequences. The chromosomal aerobactin region showed a high degree of conservation when compared with the homologous region in plasmid pColV-K30, although it was located on a different restriction endonuclease site environment.
Analyses of gonococcal lipopolysaccharide in whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: stable association of lipopolysaccharide with the major outer membrane protein (protein I) of Neisseria gonorrhoeae
Authors: Hitchcock P J
Published: January 2020

The lipopolysaccharide (LPS) of Neisseria gonorrhoeae whole-cell lysates and proteinase K-digested lysates was examined and compared with purified homologous LPS by a method which preferentially stains LPS in polyacrylamide gels. The silver-stained profile of gonococcal LPS in the proteinase K-digested lysate was similar to that of homologous purified LPS; however, the LPS profile in whole-cell lysates was much smaller than that of digested lysates or purified LPS. Conditions of solubilization did not affect these differences. Since it is known that LPS migrates in a unique fashion in second-dimension electrophoresis, the location of LPS in the whole-cell lysates was probed by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a variety of stains and radiolabels. Results from these experiments indicated a stable and reproducible association of LPS with proteins ranging between 23,000 to 36,000 in Mr, in particular major outer membrane protein I. In addition to staining with the silver method, which preferentially stains LPS, the putative LPS was resistant to digestion by proteinase K, did not stain with Coomassie brilliant blue, and was not labeled extrinsically with 125I (Iodogen method) or intrinsically with [35S]methionine. Analysis of two-dimensional gels by immunoblotting with rabbit antisera prepared from protein I bands removed from a polyacrylamide gel revealed the presence of antigens in the same area of the gel (below proteins that were 23,000 to 36,000 in Mr). Antibodies to constituents which migrated below the diagonal were essentially removed by adsorption of antisera with purified LPS, as were antibodies to homologous LPS and LPS in proteinase K-digested whole-cell lysates. Immunoblotting with a monoclonal antibody specific for LPS demonstrated reactivity of the antibody with LPS and with the protein I band. On the basis of these data, we conclude that protein I and perhaps other proteins in the whole-cell lysate are stably associated with LPS; this complex is resistant to dissociation in sodium dodecyl sulfate at high temperature (approximately 100 degrees C) but does, for unknown reasons, dissociate with electrophoresis in the second dimension. The association of LPS with protein antigens in sodium dodecyl sulfate-polyacrylamide gels adds another dimension of complexity to analysis of these antigens by immunoelectroblotting. Furthermore, the tight association of LPS with the major outer membrane protein I may alter the nature of the immune response generated by "purified" protein I vaccine antigens. The possible role of protein-LPS complexes in the pathogenesis of gonorrhea is discussed.
Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis
Authors: Ernst J D , Hartiala K T , Goldstein I M , Sande M A
Published: January 2020

Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection.
Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile
Authors: Rothman S W , Brown J E , Diecidue A , Foret D A
Published: January 2020

Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.
Loss of pili and decreased attachment to human cells by Neisseria meningitidis and Neisseria gonorrhoeae exposed to subinhibitory concentrations of antibiotics
Authors: Stephens D S , Krebs J W , McGee Z A
Published: January 2020

Recent evidence has suggested that surface structures of pathogenic bacteria, which are important in attachment to human mucosal surfaces, may be absent on bacteria grown in the presence of subinhibitory concentrations of antibiotics. We studied the effect of tetracycline and penicillin on meningococcal and gonococcal pili. Subinhibitory concentrations of tetracycline and penicillin were found to markedly reduce the number of pili per meningococcus or gonococcus and the percentage of meningococci or gonococci with pili, as determined by negative-staining electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations suggested that tetracycline decreased expression of pili by inhibiting synthesis of pilin subunits. In contrast, pilin subunit synthesis was unaltered by penicillin, suggesting a defect in assembly of pilin subunits or in anchoring of assembled pili. The decrease in the number of pili that occurred with subinhibitory concentrations of both tetracycline and penicillin was accompanied by a marked decrease in the ability of the organisms to attach to human cells. Gonococci or meningococci removed from the influence of subinhibitory concentrations of the antibiotics regained piliation, and attachment returned to levels near those of controls. The expression of meningococcal and gonococcal pili may be affected by factors that influence synthesis of pilin subunits or factors that interfere with the assembly and anchoring of pili in the outer membrane.
Hemolytic factors in Schistosoma japonicum eggs
Authors: Asahi H , Moribayashi A , Sendo F , Kobayakawa T
Published: January 2020

Extracts of Schistosoma japonicum eggs were found to exhibit hemolytic activity on erythrocytes of various species. The hemolytic reaction took place more rapidly at 37 degrees C than at 4 degrees C and did not require divalent cations. The degree of hemolysis was dependent on the concentration of the egg extracts. The hemolytic factors seemed to be lipid in nature because of being heat-stable and soluble in chloroform solvent. Further analysis by means of thin-layer chromatography showed that hemolytic activity in the extracts was due to free fatty acids. Analysis by gas chromatography revealed that fatty acids in the egg extracts consist of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids. Among them, based on the experiments with commercially available fatty acids, arachidonic acid was the most intensively hemolytic, followed by linoleic, linolenic, oleic, palmitoleic, and myristic acids in that order, whereas palmitic acid showed weak activity only in the presence of divalent cations. From the estimated calculation, it was assumed that 1 mg of protein of the egg extracts contains as much as 0.22 mg of free fatty acids.
Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin
Authors: Smit H , Gaastra W , Kamerling J P , Vliegenthart J F , de Graaf F K
Published: January 2020

The erythrocyte receptor for Escherichia coli K99 fimbrial adhesin was isolated from equine erythrocytes and characterized as Neu5Gc-alpha(2----3)-Galp-beta(1----4)-GLcp-beta(1----1)-Ceramide. This glycolipid acted as the receptor for K99 by four different experimental approaches: inhibition of equine erythrocyte hemagglutination by preincubation of K99-positive bacteria or purified K99 fimbriae with the isolated glycolipid; inhibition of attachment of K99-positive bacteria to porcine intestinal epithelial cells in the presence of the isolated glycolipid; induction of binding of K99-positive bacteria or purified K99 fimbriae to normally unreactive guinea pig erythrocytes by coating these cells with the isolated glycolipid; and isolation of the receptor by affinity chromatography with K99 coupled to CNBr-activated Sepharose 4B, indicating a strong interaction between K99 and the isolated glycolipid.
Human phagocyte interactions with the Lyme disease spirochete
Authors: Peterson P K , Clawson C C , Lee D A , Garlich D J , Quie P G , Johnson R C
Published: January 2020

The interaction between human polymorphonuclear leukocytes and monocytes and the Lyme disease spirochete was investigated by incubating phagocytes with microorganisms adherent to plastic or glass surfaces. Both cell populations readily phagocytized and killed spirochetes, and antibodies facilitated but were not essential for phagocytosis.
Comparative superoxide-generating system of granulocytes from blood and peritoneal exudates
Authors: Zimmerli W , Lew P D , Cohen H J , Waldvogel F A
Published: January 2020

Blood polymorphonuclear leukocytes (PMN), upon interaction with specific chemotactic stimuli, leave the blood stream and migrate to tissues. At such a location, and upon contact with invading microorganisms, they generate superoxide (O2-) as a part of the respiratory burst of phagocytosis. We have compared the O2(-)-generating system of guinea pig peritoneal exudate PMN with that of peripheral blood PMN from the same species or of human origin. The rate of O2- production by casein-induced guinea pig exudate cells in response to a particulate stimulus (opsonized zymosan) was significantly decreased when compared with peripheral blood PMN. Furthermore, the activation time of the O2(-)-generating system was shorter in exudate than in peripheral blood PMN. Important differences in sensitivity to a metabolic inhibitor were found: (i) N-ethylmaleimide increased the activation time of the O2(-)-generating system only in guinea pig exudate cells and not in blood cells; (ii) when N-ethylmaleimide was added after complete activation of the PMN, the O2- production rate was inhibited in guinea pig as well as human blood PMN, but not in guinea pig exudate PMN. We conclude that exudation markedly alters one of the most important antibacterial mechanisms of PMN, the superoxide-generating system.
Metabolic and mitochondrial morphological changes that mimic Reye syndrome after endotoxin administration to rats
Authors: Yoder M C , Egler J M , Yudkoff M , Chatten J , Douglas S D , Polin R A
Published: January 2020

The administration of sublethal doses of Escherichia coli O111:B4 endotoxin to starved rats results in significant increases in plasma ammonia, free fatty acids, and serum lactate compared with starved controls. These metabolic alterations are associated with Reye syndrome-like histological findings of hepatic microvesicular fatty accumulation and hepatic ultrastructural evidence of mitochondrial pleomorphism with matrix disruption. This sublethal endotoxin model may help elucidate the relationship between the hepatic mitochondrial injury, characteristic metabolic impairment, and encephalopathy seen in patients with Reye syndrome.
Identification of protein antigens of Legionella pneumophila serogroup 1
Authors: Pearlman E , Engleberg N C , Eisenstein B I
Published: January 2020

Growth of Legionella pneumophila serogroup 1 in yeast extract broth was standardized, and protein profiles of detergent-solubilized cells were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Over 30 protein bands were identified, 6 of which were more prominent both in Coomassie brilliant blue-stained profiles and in fluorograms with intrinsically radiolabeled bacteria. These major proteins were 22,000 dalton (22K), 24K, 43K, 63K, 76K, and 78K. We found that the 24K and 63K major proteins were antigenic, as demonstrated both by radioimmunoprecipitation (RIP) of [35S]methionine-labeled organisms and by immunoblotting with rabbit hyperimmune sera. In addition, both techniques detected antigens migrating at 58K, 72K, 76K, and 78K. The major 22K and 43K major proteins and antigens migrating at 25.5K, 29K, and 46K were only detected by radioimmunoprecipitation, whereas antigens of 19K, 48K, 53K, and 68K were detected only by immunoblotting. Cell-surface localization of the proteins was determined by a modified radioimmunoprecipitation assay designed to react specifically with surface antigens and by the use of hyperimmune sera that had been extensively preabsorbed with intact cells to deplete the sera of antibodies directed against surface components. The 22K, 24K, 43K, 63K, and 78K major proteins and several minor proteins were found to be located on the surface of L. pneumophila cells.
Purification and characterization of a receptor for the 987P pilus of Escherichia coli
Authors: Dean E A , Isaacson R E
Published: January 2020

A receptor-containing fraction that caused visible aggregation of piliated Escherichia coli strain 987 bacteria was released into solution when brush borders from adult rabbit small intestines were stored. The receptor-containing fraction was separated into more than 50 bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 4 of which contained carbohydrate as demonstrated by staining with periodic acid-Schiff. Receptor activity in the receptor-containing fraction was associated with a low-molecular-weight (less than 14,000) glycoprotein as measured by Western blots. Extraction of brush borders with chloroform-methanol-water (4:8:3) resulted in increased pilus 987P receptor activity in the aqueous extract. Gel filtration chromatography of sodium dodecyl sulfate-solubilized aqueous extract yielded a fraction with 987P receptor activity that contained only the low-molecular-weight glycoprotein. The purified 987P receptor contained 81% carbohydrate and 19% amino acids by weight. Isoelectric focusing separated the 987P receptor receptor into 3 peaks corresponding to isoelectric points of 2.2, 3.8, and 4.1. Each of the peaks contained 987P receptor activity. The 987P receptor activity was sensitive to periodate oxidation and to digestion with pronase. The strain 987-agglutinating activity of the receptor was inhibited by amino sugars and their N-acetylated derivatives, by compounds having a free amino group, by high concentrations of salt, and by lectins that recognize D-galactose or L-fucose.
Differences in expression and exposure of promastigote and amastigote membrane molecules in Leishmania tropica
Authors: Sadick M D , Raff H V
Published: January 2020

The accessibility of particular Leishmania tropica promastigote (extracellular) and amastigote (intracellular) membrane molecules might be related to the relative abilities of the two stages to induce host immune responses. To examine the exposure of membrane antigens on resident macrophage-susceptible promastigotes and resident macrophage-resistant amastigotes, both stages were analyzed by polyacrylamide gel electrophoresis and immunoblotting after specific labeling and extraction procedures. Protein compositional studies, using metabolic labeling of promastigotes and amastigotes, demonstrated that both forms possessed numerous endogenously synthesized proteins. In addition, a marked difference was revealed in the external exposure of promastigote and amastigote membrane constituents when analyzed by 125I surface labeling or Western blot analysis. Whereas nine promastigote proteins were intensely to moderately iodinated, only one amastigote membrane component was similarly labeled (9.5K band). Western blot analyses with serum from a rabbit immunized with a mixture of both L. tropica stages indicated that the majority of promastigote molecules accessible to 125I may also react with immune serum. However, Western blots of extracted amastigotes identified several bands not seen on radiographs and thus not accessible to 125I. The external exposure of these amastigote molecules was confirmed in that immune serum adsorbed with viable, intact amastigotes was no longer reactive with amastigote extracts. Further, by Western blot analyses of sodium dodecyl sulfate- but not Nonidet P-40-extracted amastigotes, three amastigote-specific membrane antigens not previously observed with nonionic detergent extraction methods were identified. The autofluorographic pattern of amastigotes intrinsically labeled with N-[3H]acetylglucosamine, an amino sugar which is incorporated into membrane carbohydrates, was in excellent agreement with the pattern of antigens reactive with antibody in Western blots. Thus, with these cell surface labeling and extraction methods, promastigote and amastigote membranes were shown to be significantly different. Amastigotes possessed several membrane-associated molecules, but few appeared to be either accessible or reactive with 125I. Moreover, the majority of molecules not reactive with 125I, but reactive with antibodies, may be glycosylated. These observations are discussed relative to the ability of amastigotes both to survive within the degradative milieu of macrophage phagolysosomes and to evade host immune reactivity.
Effect of toxic shock syndrome toxin 1 on chicken embryos
Authors: de Azavedo J C , Lucken R N , Arbuthnott J P
Published: January 2020

Staphylococcus aureus strains associated with toxic shock syndrome produce toxic shock syndrome toxin 1 (TSST1). This toxin has a variety of biological effects, including enhanced lethality in rabbits in the presence of sublethal amounts of lipopolysaccharide (LPS). Because chicken embryos are highly susceptible to LPS, the synergistic effect of TSST1 and LPS was examined in this system. Although TSST1 per se had no effect on chicken embryos, it potentiated the lethal effect of LPS. The 50% lethal dose of LPS was greatly reduced in the presence of up to 10 micrograms of TSST1 per ml. However, at high doses of TSST1 (greater than 100 micrograms/ml), no enhanced lethality was observed. The lowest dose of TSST1 tested which potentiated lethality was 10 ng/ml.
Cellular and extracellular protein antigens of Treponema pallidum synthesized during in vitro incubation of freshly extracted organisms
Authors: Stamm L V , Bassford P J
Published: January 2020

A new medium that permits radiolabeling of freshly extracted cells of Treponema pallidum with [35S]methionine very efficiently has been devised. Although treponemes were not purified free of contaminating rabbit tissue, label was incorporated exclusively into treponemal protein in a linear manner for at least the first 16 h of in vitro incubation. Throughout this period, virtually a full complement of treponemal proteins was synthesized, based on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparison of the radiolabeled protein profile with the Coomassie blue-stained profile of gradient-purified treponemes. The radiolabeled protein profiles obtained with three pathogenic strains were very similar but not identical. Using solubilized treponemal extracts and a sensitive radioimmunoprecipitation procedure, we identified the protein antigens of T. pallidum that were recognized by immunoglobulin G antibodies in various rabbit and human syphilitic sera. A simple fractionation procedure has been used to separate soluble and membrane-bound treponemal proteins. A number of the membrane proteins are exposed on the cell surface, since intact radiolabeled treponemes bound antibodies directed against these proteins. In addition, a unique class of low-molecular-weight extracellular treponemal proteins has been identified. The cell surface-exposed proteins were among the earliest proteins recognized by immunoglobulin G antibodies after experimental infection of rabbits with T. pallidum.
Competitive displacement of mutans streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R
Authors: Tanzer J M , Kurasz A B , Clive J
Published: January 2020

The ability of Streptococcus salivarius TOVE-R to displace virulent representatives of the most prevalent human mutans streptococci from the teeth of rats, and thereby to inhibit caries, was studied. Streptococcus mutans 10449S- or Streptococcus sobrinus 6715-13WT-infected specific-pathogen-free rats consuming a high-sucrose diet were inoculated by TOVE-R. The infectants were differentially recovered from swabs of the teeth over the time course of infection and from sonically treated material of extracted teeth and excised tongues. Despite initial colonization of the teeth by the mutans streptococci, TOVE-R colonized the teeth, unlike other essentially nonvirulent plaque formers already described. It did not colonize the tongues of the rats. TOVE-R emerged and persisted as a prominent member of the plaque ecology. There was an associated decline in the mutans streptococci on the teeth, and this decline was associated with significant inhibition of the caries component attributable to 10449S infection (56%) and to 6715-13WT infection (52%). TOVE-R did not reliably inhibit the component of fissure caries attributable to the nonmutans indigenous flora of the rats. TOVE-R itself induced no detectable decay. The data suggest the potential therapeutic utility of TOVE-R to inhibit caries by displacement of mutans streptococci from the teeth. These results supplement the already reported ability of TOVE-R to preempt initial colonization of teeth by the mutans streptococci.
Immunochemistry of the Streptococcus mutans BHT cell membrane: detection of determinants cross-reactive with human heart tissue
Authors: Ayakawa G Y , Siegel J L , Crowley P J , Bleiweis A S
Published: January 2020

Cell membranes of Streptococcus mutans BHT serotype b were prepared after glass bead disruption or mutanolysin digestion of whole cells. Immunoblot analyses of BHT membrane extracts revealed major polypeptides of 42,000, 46,000, 62,000, and 82,000 daltons, as well as several minor bands, to be reactive with rabbit anti-human heart immunoglobulins. Heart cross-reactive antigens have been reported in the cell walls and culture fluids of several S. mutans serotypes. This represents the first report of cell membrane-localized heart cross-reactive antigens in this oral pathogen. Positive enzyme-linked immunosorbent assay and immunoblot reactions were also obtained with heart tissue antigen and anti-BHT sera, indicating mutual cross-reactivity. The major cross-reactive component detected by immunoblotting of human heart extracts was a 69,000-dalton polypeptide.
Use of auxotyping for epidemiological studies of Campylobacter jejuni and Campylobacter coli infections
Authors: Tenover F C , Knapp J S , Patton C , Plorde J J
Published: January 2020

A chemically defined medium developed for Neisseria gonorrhoeae was modified to support the growth of Campylobacter jejuni and Campylobacter coli. A total of 76 isolates of C. jejuni and 14 isolates of C. coli were tested on this medium, which was designated Campylobacter defined medium (CDM), over a 3-month period. Although none of the C. coli isolates appeared to require amino acids, 51% of the C. jejuni tested required one and 7% required multiple amino acids for growth. An analysis of isolates obtained from three household outbreaks of campylobacteriosis demonstrated that auxotyping identified the epidemic strain within each outbreak. Among 70 isolates of C. jejuni examined, no correlation could be drawn between a specific serotype and auxotype or between auxotype and plasmid profile.