bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of urease gene sequences from Providencia stuartii
Authors: Mobley H L , Jones B D , Jerse A E
Published: January 2020

Providencia stuartii was the most prevalent isolate recovered from urine specimens taken weekly over a 1-year period from 51 nursing home patients with urinary catheters in place. Thirty percent of the isolates were urease positive. Urease, which is implicated in renal stone formation, was shown to be transmissible on an 82-kilobase conjugative plasmid in one isolate. Plasmid DNA isolated from this strain was digested with EcoRI, ligated into the EcoRI site of pBR322, and used to transform Escherichia coli HB101. Ampicillin-resistant clones were replica plated onto urea segregation agar, and a urease-positive clone, designated pMID101, was isolated. Recombinant and native urease from cell lysates had identical electrophoretic mobilities on nondenaturing polyacrylamide urease activity gels. The native enzyme was induced fourfold when cells were grown in the presence of 0.1% urea and had a km of 9.4 mM and a Vmax of 3.2 mumol of NH3 per min per mg of protein. Its molecular weight was estimated to be 375,000 +/- 35,000 by Sephacryl S-300 chromatography. The enzyme was cytoplasmic in P. stuartii, was inhibited in vitro by hydroxyurea, acetohydroxamic acid, and EDTA, and appears to have a complex subunit structure and a unique molecular size within genera of the Proteeae tribe.
Transfer of the plasmid for exfoliative toxin B synthesis in mixed cultures on nitrocellulose membranes
Authors: Rogolsky M , Beall B W , Wiley B B
Published: January 2020

Plasmid pRW002 carries genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis. When a donor strain carrying plasmid pRW002 was mixed with a plasmidless recipient strain on a nitrocellulose membrane in accordance with the procedure used for staphylococcal conjugation, pRW002 was passed to the recipient by mixed-culture transduction. Transfer was inhibited by citrate and serotype B phage antisera but not by DNase I. Cell-to-cell contact was not required, and transfer frequencies increased more than 10-fold in the presence of small concentrations of mitomycin C. These results are consistent with pRW002 transfer in mixed cultures by transduction and not by conjugation or transformation. Immunodiffusion and DNA analyses after agarose gel electrophoresis demonstrated that transductants were exfoliative toxin B producers and housed pRW002. Since mixed-culture transfer has been reported to occur on skin, our results suggest that mixed-culture transduction might be a mechanism for the transfer of genetic determinants for pathogenicity in vivo.
Surface proteins of Bordetella pertussis: comparison of virulent and avirulent strains and effects of phenotypic modulation
Authors: Armstrong S K , Parker C D
Published: January 2020

The surface proteins of several Bordetella strains and their modulated derivatives were examined by surface radioiodination, cell fractionation, and Western blotting. A surface protein with a high Mr, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis and Bordetella parapertussis cells and was absent in avirulent B. pertussis strains. The electrophoretic profiles of lipopolysaccharide and the 40,000-Mr anion-selective porin were not determinants which correlated with phase variation or phenotypic modulation. At least three envelope proteins (91,000, 32,000, and 30,000 molecular weight) were found only in virulent B. pertussis strains and were absent or diminished in the avirulent phase and most phenotypically modulated strains. Two transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits.
Evidence for N-terminal exposure of the protein IA subclass of Neisseria gonorrhoeae protein I
Authors: Judd R C
Published: January 2020

The JS3 and FA638 strains of Neisseria gonorrhoeae bear a protein IA subclass of protein I (P.I). The purified P.Is of surface-labeled strains JS3 and FA638 were cleaved with the N-terminal degradation enzyme leucine amino peptidase (LAP), and the resultant fragments were separated in sodium dodecyl sulfate-polyacrylamide gels. Autoradiography demonstrated that the surface radiolabel was absent in a LAP-generated P.I peptide that was about 1,900 daltons lower in apparent molecular mass than the native P.I in both strains. Moreover, the 4G5 monoclonal epitope, known to be located on the surface of the organism, was also absent in the LAP-generated P.I peptide that was about 1,900 daltons less in apparent molecular mass than the original P.I of strain FA638. These data strongly suggest that the N terminus of the P.IA subclass is exposed on the surface of the bacterium and that this region represents about 5%, or 15 to 20 amino acids, of the total protein.
Inability of pyrogenic, purified Bordetella pertussis lipid A to induce interleukin-1 release by human monocytes
Authors: Caroff M , Cavaillon J M , Fitting C , Haeffner-Cavaillon N
Published: January 2020

Free lipid A of Bordetella pertussis, Neisseria meningitidis, and Escherichia coli lipopolysaccharide (LPS) was prepared by hydrolysis in acetate buffer (pH 4.5); in addition, lipid A from B. pertussis and E. coli was prepared by hydrolysis in mineral acid (HCl). The precipitates obtained were purified by extraction methods in toluene-methanol and are referred to as crude lipid A. Purified lipid A from N. meningitidis and B. pertussis was obtained by extraction in a mixture of chloroform-methanol-water-triethylamine. The different preparations were tested for their pyrogenicity (endogenous pyrogen; EP) and their capacity to trigger the release of interleukin-1 (IL-1; previously known as lymphocyte-activating factor; LAF) by human monocytes. Crude lipid A from E. coli and N. meningitidis were both IL-1 inducers. Crude B. pertussis lipid A (acetate buffer; pH 4.5), which contains a beta-1-6-linked D-glucosamine disaccharide, two phosphoryl groups, and five fatty acids, was pyrogenic and an IL-1 inducer (EP+/LAF+); but crude B. pertussis lipid A (0.25 N HCl), which lacked the glycosidic phosphoryl group, was 1,000-fold less pyrogenic than the diphosphorylated lipid A, yet it retained its IL-1-inducing capacity (EP-/LAF+). Purified N. meningitidis lipid A was not an inducer of IL-1 release and purified B. pertussis lipid A exhibited identical pyrogenicity as the parent LPS but was devoid of any IL-1-release inducing capacity (EP+/LAF-). These results demonstrate that for some endotoxins, purified lipid A is unable to induce IL-1 release by human monocytes; however, it is pyrogenic, supporting the hypothesis that IL-1 and EP are induced by different determinants on the LPS molecule.
Modulation of Actinomyces viscosus colonization of mouse teeth in vivo by immunization with fimbrial adhesins
Authors: Crawford P C , Clark W B
Published: January 2020

Experiments were performed to determine whether immunization of mice with fimbrial adhesins isolated from Actinomyces viscosus T14V could modulate infection of tooth surfaces in animals challenged with the homologous strain. Saliva and sera from animals immunized in the submandibular gland region contained elevated levels of fimbria-specific immunoglobulin A (IgA) and IgG, whereas saliva and sera from sham-immunized animals did not. There was a statistically significant inverse correlation between the presence of fimbria-specific antibodies in saliva and serum and the levels of bacterial colonization on molar tooth surfaces. These results suggest that fimbrial adhesins may effectively modulate infection of tooth surfaces by periodontopathic bacteria.
An 88,000-Mr Giardia lamblia surface protein which is immunogenic in humans
Authors: Edson C M , Farthing M J , Thorley-Lawson D A , Keusch G T
Published: January 2020

Human anti-Giardia lamblia sera specifically immunoprecipitated an 88,000-Mr surface protein from radioiodinated trophozoites, establishing this protein as a potentially important immunogen in humans. A mouse monoclonal antibody (GL-1) was isolated which immunoprecipitated the same 88,000-Mr surface protein recognized by the human sera. GL-1 gave uniform fluorescent staining of the cell surface and flagella of G. lamblia trophozoites from the Portland 1 and WB strains as well as fresh clinical isolates, but not of Giardia muris, suggesting that the surface antigen is specific to G. lamblia. Other human parasites, including Entamoeba histolytica, Trichomonas vaginalis, and Trichomonas hominis, were not stained. A second mouse monoclonal antibody (GL-2) gave weaker immunofluorescent staining of living G. lamblia trophozoites but intense staining of fixed cells. None of the other parasites tested were stained, with the exception of E. histolytica, which may contain a cross-reactive antigen. No proteins were recognized in immunoprecipitation studies with iodinated trophozoites.
Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O
Authors: Hugo F , Reichwein J , Arvand M , Krämer S , Bhakdi S
Published: January 2020

Murine monoclonal antibodies were generated against streptolysin O. One out of 10 tested immunoglobulin clones exhibited strong neutralizing activity; in solution, the presence of approximately two to four antibody molecules per toxin monomer effected 50% neutralization of hemolytic toxin activity. An enzyme-linked immunosorbent assay performed with target cell membranes that were treated with streptolysin O in the presence and absence of neutralizing antibodies showed that the antibodies did not block primary binding of the toxin to the cells. When membranes were solubilized in deoxycholate detergent and centrifuged in sucrose density gradients, those lysed with streptolysin O contained detergent-resistant, high-molecular-weight oligomers identical to the pore lesions, whereas those given toxin and neutralizing antibody contained the toxin exclusively in low-molecular-weight, nonoligomerized form. The process of pore formation by streptolysin O must thus involve two distinct steps, i.e., the primary binding of toxin molecules to the membrane followed by oligomerization of bound toxin monomers by lateral aggregation in the lipid bilayer to form the transmembrane pores.
Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones
Authors: Deepe G S , Smith J G , Sonnenfeld G , Denman D , Bullock W E
Published: January 2020

Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells.
Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes
Authors: Havell E A
Published: January 2020

Listeria monocytogenes, a gram-positive facultative intracellular bacterium, was shown to be capable of infecting and proliferating in murine embryo fibroblasts. During exponential proliferation, the doubling time of the bacterium was determined to be 2.5 h intracellularly, compared with 25 min extracellularly. Progressive intracellular growth of listeriae ultimately resulted in the destruction of initially infected cells and the spread of infection to neighboring cells. Listeria infection induced fibroblasts to synthesize considerable quantities of an acid-stable interferon that proved to be antigenically indistinguishable from both polyinosinic-polycytidylic acid-induced and virus-induced interferon.
Surface-exposed antigenic cleavage fragments of Neisseria gonorrhoeae proteins 1A and IB
Authors: Schmitt S , Layh G , Buchanan T B
Published: January 2020

Whole bacteria, isolated outer membranes, and purified protein I (PI) from one transparent (O-) and two different opaque (O+) phenotype gonococcal strains (serogroups I, II, and III; PI serotypes 1, 5, and 9b) were each treated with tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin, alpha-chymotrypsin, and proteinase K. Protein IA (PIA) of strain 7122 (O-, serotype 1, serogroup I) was resistant to proteolysis by tolysulfonyl phenylalanyl chloromethyl ketone-trypsin and alpha-chymotrypsin and only slightly affected by proteinase K, as long as it was associated with intact bacteria or isolated outer membranes. Purified PIA however was cleaved by these enzymes, resulting in two to five fragments. In contrast, all preparations of strains 5766 opaque phenotype (O+, serotype 7, serogroup II) and 1955 (O+, serotype 9b, serogroup III) were accessible to proteolysis, resulting in cleavage fragments of PIB compatible to those described previously by O. Barrera and J. Swanson (Infect. Immun. 44:565-568, 1984), M. S. Blake et al. (Infect. Immun. 33:212-222, 1981), and Blake (in G. K. Schoolnik, ed., The Pathogenic Neisseriae, 1985). Our data indicated that the purified PIB fraction was more accessible to proteases than the PIBs of whole bacteria or outer membranes. The fragmentation pattern of PIA cleavage products were quite different from PIB fragments, consistent with the different structure of these two groups of PI molecules. Time-dependent cleavage experiments with proteases, i.e., alpha-chymotrypsin, indicated that PIA was subsequently cleaved into smaller fragments. Highly reactive monoclonal antibodies, each specific for a surface-exposed epitope of PIA of strain 7122 or PIB of strains 5766 and 1955, as assessed by coagglutination, Western blot, and immunofluorescence, were reacted with PIA and PIB cleavage fragments in Western blot experiments. All cleavage fragments of the purified PIA and PIB preparations with molecular weights of greater than or equal to 14,200 showed immune reaction in Western blotting, whereas whole cell and outer membrane PIB fragments were less reactive with the specific monoclonal antibodies.
Ultrastructural study of mode of entry of Chlamydia psittaci into L-929 cells
Authors: Hodinka R L , Wyrick P B
Published: January 2020

The entry of Chlamydia psittaci into L-929 cells was studied morphologically by transmission electron microscopy and quantitatively by a method that discriminates between attachment and uptake. Upon adsorption of 3H-labeled elementary bodies (EBs) to host cells at 4 degrees C, the EBs bound efficiently to the L-cell surface. Binding reached an equilibrium level of 55% in 3 h. Ultrastructural analysis revealed that EBs were bound preferentially to the tips and sides of microvilli at this temperature. The EBs were also observed in coated pits located at the bases of microvilli and along smooth surfaces of the host cell. No internalization was observed at 4 degrees C. When cells with prebound 3H-labeled EBs were warmed to 37 degrees C, the EBs rapidly became resistant to proteinase K removal (half time = 5 min), indicating ingested chlamydiae. At 37 degrees C, the EBs were internalized within tightly bound vesicles surrounded by an electron-dense coat of fibrillar material. EBs were also present in smooth-surfaced pits and vesicles of the host cell. Using alpha 2-macroglobulin coupled to colloidal gold (a known marker for receptor-mediated endocytosis), we observed that the entry of EBs into cells via coated pits was identical in appearance to the internalization of alpha 2-macroglobulin. Also, when the two ligands were mixed together, they could be seen within the same coated pits and were cointernalized within endocytic vesicles of the host cell. These results suggest that C. psittaci can enter nonprofessional phagocytic cells by a pathway which is similar to that of receptor-mediated endocytosis of many physiologically important macromolecules, bacterial toxins, and viruses.
Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis
Authors: Tuomanen E
Published: January 2020

Two proteins secreted by Bordetella pertussis are known to mediate adherence of these bacteria to mammalian respiratory cilia. When either ciliated cells or other pathogenic bacteria were pretreated with these adhesins, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus acquired the ability to adhere to cilia in vitro and in vivo. Such piracy of adhesins may contribute to superinfection in mucosal diseases such as whooping cough.
Modulation of lysosomal protease-esterase and lysozyme in Kupffer cells and peritoneal macrophages infected with Nocardia asteroides
Authors: Black C M , Paliescheskey M , Beaman B L , Donovan R M , Goldstein E
Published: January 2020

Virulent Nocardia asteroides reduces lysosomal acid phosphatase activity in murine macrophages. A computer-assisted imaging photometry system was used to quantitate lysozyme and nonspecific esterase-neutral protease levels within individual macrophages following ingestion of nocardiae. In contrast to acid phosphatase, lysozyme and esterase-neutral protease activity was either unchanged or increased following infection by increasing numbers of nocardial cells.
Cross-reactions of lipopolysaccharides of Pseudomonas aeruginosa in antipneumococcal and other antisera
Authors: Heidelberger M , Horton D , Haskell T H
Published: January 2020

Lipopolysaccharides of the seven Fisher immunotypes of Pseudomonas aeruginosa gave cross-precipitation in many antipneumococcal sera. The reaction of Pseudomonas type IV in type 25 antipneumococcal serum was immediate and heavy: 93 micrograms of antibody nitrogen per ml. Correlations are described, mainly between the structures of the O-chains of the immunotypes and their specificities as shown by the cross-reactions.
Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin
Authors: Suttorp N , Seeger W , Zucker-Reimann J , Roka L , Bhakdi S
Published: January 2020

The effects of staphylococcal alpha-toxin on arachidonic acid metabolism in rabbit polymorphonuclear leukocytes (PMNs) were investigated and compared with those of the ionophore A23187 and the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLP). Sublytic amounts of alpha-toxin stimulated the release of leukotriene B4 (LTB4) in PMNs in a dose-dependent manner. The toxin was several times more potent than fMLP but was not as effective as the ionophore. Preincubation of the toxin with neutralizing antibodies abolished the effect. Extracellular calcium was strictly required for eliciting LTB4 generation. Verapamil, a calcium channel blocker, inhibited fMLP-mediated LTB4 generation but had no effect on alpha-toxin- or A23187-exposed PMNs. Agents such as trifluoperazine and N-6(aminohexyl)-5-chloro-1-naphthalene sulfonamid that interfered with calmodulin activity, however, inhibited LTB4 generation in all cases. One minute after the addition of alpha-toxin, PMNs exhibited a severalfold enhancement in passive permeability to 45Ca2+. In addition, these cells became permeable to sucrose but not to inulin or dextran. The influx pattern was consistent with the previous observation that alpha-toxin creates discrete transmembrane channels in erythrocytes with an effective internal diameter of 2 to 3 nm. The results suggest that alpha-toxin triggers the arachidonic acid pathway in PMNs by facilitating calcium influx into the cells, possibly via transmembrane toxin pores that serve as calcium gates. Generation of arachidonic acid metabolites in PMNs by sublytic amounts of alpha-toxin may represent an important cellular reaction that generally occurs during infections with Staphylococcus aureus.
Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli
Authors: Brown D R , Parker C D
Published: January 2020

Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392. One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B. pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA. FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E. coli, was larger than that expressed in B. pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria. The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases. The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene. Conjugative transfer of pDB441 to B. pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B. pertussis.
A murine monoclonal antibody specific for the outer core oligosaccharide of Salmonella lipopolysaccharide
Authors: Tsang R S , Chan K H , Chau P Y , Wan K C , Ng M H , Schlecht S
Published: January 2020

Immunoglobulin G3 murine monoclonal antibody T6 specific for the lipopolysaccharide of Salmonella O serogroups A to E was established. By using R mutants of Salmonella spp., Escherichia coli, and Shigella spp., the major reactive epitope with T6 was tentatively identified as the terminal disaccharide, N-acetylglucosamine 1.2----alpha glucose, of the core oligosaccharide. T6 was reactive with 10 clinical isolates of each of the Salmonella O serogroups A to E but not with 58 isolates of other gram-negative bacteria. Its selective reactivity against Salmonella spp. renders T6 a potentially more useful reagent than the conventional polyvalent serum for the identification of Salmonella spp. It may also serve as a useful molecular tool for the study of the outer core structure of all Salmonella and related species.
Rickettsia prowazekii requires host cell serine and glycine for growth
Authors: Austin F E , Turco J , Winkler H H
Published: January 2020

The growth requirement of Rickettsia prowazekii for the amino acids serine and glycine was assessed in both wild-type cell lines and a mutant cell line. X-irradiated L929 cells supported the growth of R. prowazekii when the cells were incubated in Eagle minimal essential medium supplemented with serum. In contrast, in this medium, X-irradiated Vero cells did not support the growth of rickettsiae unless cycloheximide, serine, or glycine was added. Other nonessential amino acids, additional glucose, and potential products of host cell metabolism of serine and glycine were nonstimulatory. The concentration of serine or glycine required to support rickettsial growth had no effect on the doubling time of uninfected, unirradiated Vero cells. A comparison of intracellular amino acid pools indicated that the serine and glycine concentrations in mock-infected Vero cells were approximately 31 and 14% of the respective concentrations in mock-infected L929 cells. The pools of both amino acids in Vero cells increased markedly upon treatment of the cells with cycloheximide. Interconversion of serine and glycine catalyzed by serine hydroxymethyltransferase was detected in cell-free extracts of purified rickettsiae. However, this enzymatic activity did not permit rickettsial growth in a glycine-requiring clone (772-56d) of the Chinese hamster ovary cell CHO-K1 in the absence of glycine supplementation. These data indicate that R. prowazekii depends on the host cell for serine or glycine.
Correlation between virulence and colony morphology in Vibrio vulnificus
Authors: Simpson L M , White V K , Zane S F , Oliver J D
Published: January 2020

Of 38 isolates of Vibrio vulnificus examined, all avirulent strains produced only translucent colonies. All virulent strains, with the exception of biogroup 2 (eel pathogens), exhibited both opaque and translucent colonies. Isogenic morphotypes were examined for a variety of phenotypic and virulence traits. Only the ability to utilize transferrin-bound iron and the presence of a surface polysaccharide were found to correlate with colony opacity and virulence.
Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin
Authors: Popoff M R
Published: January 2020

Lethal toxin (LT) was purified from Clostridium sordellii IP82 by DEAE-Trisacryl, Ultrogel AcA3-4 gel filtration, and hydroxyapatite column chromatography. The molecular weight of purified LT was estimated to be 240,000 to 250,000, and the pI was at pH 4.55. LT was lethal for mice by intraperitoneal injection (3.4 X 10(5) mouse lethal doses per mg of protein), cytotoxic for Vero cells (6.1 X 10(4) cytotoxic units per mg of protein), erythematous and edematous by intradermal injection in guinea pigs, and induced a moderate fluid accumulation in the guinea pig intestinal loop test. The lethal activity was inactivated by N-bromosuccinimide, N-chlorosuccinimide, chloramine-T, and sodium dodecyl sulfate. The data suggest that tryptophan and methionine residues present in the toxin are important for lethal activity. Furthermore, LT was inactivated by oxidized glutathione and activated by dithiothreitol. Inactivation by sulfhydryl-group reagents 5,5'-dithiobis(2-nitrobenzoic acid) and iodoacetamide was only obtained with dithiothreitol-treated LT. Thiol groups which are protected as a disulfide bond(s) seem to be essential for the LT activity. A specific antiserum against LT neutralized the biological activities of LT and also cytotoxic activity and lethal activity of Clostridium difficile toxin B but not of C. difficile toxin A. However, this serum did not recognize antigen from C. difficile culture supernatant by immunoblotting. It was concluded that antibodies prepared from C. sordellii LT that neutralized C. difficile cytotoxic activity recognized a low number of epitopes or tertiary structures of C. difficile cytotoxin.
Production of an extracellular toxic complex by various strains of Klebsiella pneumoniae
Authors: Straus D C
Published: January 2020

Six isolates of Klebsiella pneumoniae (two serotype 1 isolates and a capsular variant of one of these, and two serotype 2 isolates and a capsular variant of one of these) possessing various degrees of virulence in rats and mice were examined for their in vitro production of an extracellular toxic complex (ETC). The ETC has been shown to be lethal for and produce extensive lung pathology in mice. This compound has been shown to be composed of capsular polysaccharide, lipopolysaccharide, and a small amount of protein. All six isolates produced the ETC. Immunization of experimental animals with sublethal doses of the ETC was protective against both homologous and heterologous strains, and this protection was due to antibody production. An examination of the various phases of growth of K. pneumoniae showed that there was extracellular release of the component parts of the ETC occurring during all phases of growth. The presence of the ETC in the supernatant fluids was due to actual release of this material as opposed to cell lysis. Antibodies to the lipopolysaccharide portion (which has been shown to possess the observed toxicity) of the ETC were protective against the homologous bacterium.
Immunosuppressive effects of Centipeda periodontii: selective cytotoxicity for lymphocytes and monocytes
Authors: Shenker B J , Berthold P , Dougherty P , Porter K K
Published: January 2020

We have examined soluble sonic extracts prepared from several strains of Centipeda periodontii for their ability to alter human lymphocyte function. These organisms were isolated from subgingival plaque of patients with periodontal disease. We found that sonicates from several, but not all, strains of C. periodontii caused a dose-dependent inhibition of lymphocyte responsiveness to concanavalin A, phytohemagglutinin, pokeweed mitogen, and formalinized Staphylococcus aureus. Inhibition was associated with a concomitant decrease in cell viability assessed by trypan blue exclusion, 51Cr release, and electron microscopy. The maximal number of dead cells was observed 20 to 24 h after exposure to the sonic extract. Susceptible cells include human lymphocytes (both B and T), monocytes, and erythrocytes, whereas polymorphonuclear cells, murine L-929 fibroblasts, and sheep erythrocytes were not affected. Preliminary characterization of the cytotoxic activity indicates that it is heat labile and trypsin sensitive and has an Mr of 60,000. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of periodontal diseases. The data presented in this paper suggest that immunosuppression (local or systemic or both) could be initiated by C. periodontii. This immunosuppression may alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of C. periodontii itself or some other opportunistic organism.
The erythrocyte and epithelial cell receptors for Haemophilus influenzae are expressed independently
Authors: van Alphen L , Poole J , Geelen L , Zanen H C
Published: January 2020

The Anton blood group antigen has been shown to be the erythrocyte receptor for Haemophilus influenzae. Cord erythrocytes, which lack the Anton antigen, were not agglutinated by H. influenzae (L. van Alphen, J. Poole, and M. Overbecke, FEMS Microbiol. Lett. 37:69-71, 1986). Twenty-eight erythrocyte suspensions from newborns less than 4 days old were also not agglutinated, but 23 of 56 erythrocyte suspensions from 4- to 50-day-old newborns and 23 of 35 erythrocyte suspensions from older infants were agglutinated. Positive hemagglutination correlated with the presence of the Anton antigen on the erythrocytes for 163 of 173 (P less than 0.0001). Adherence of H. influenzae to buccal epithelial cells obtained from six newborns within 3 days after birth was as strong as that found with adult epithelial cells, whereas the erythrocytes from five of six of these newborns were not agglutinated by the bacteria. Adherence of H. influenzae to epithelial cells of 15 donors was not inhibited by anti-Anton serum. Moreover, H. influenzae carrying fimbriae adhered to epithelial cells of an Anton-negative donor. From these results we conclude that the age at which the erythrocyte receptor for H. influenzae is expressed is the same as for the Anton antigen, but that the receptor on the epithelial cells is already expressed at birth and is not identical to the Anton antigen.
Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay
Authors: Schwarz S , Ellen R P , Grove D A
Published: January 2020

There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of 3H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.
Nonoxidative antimicrobial effects of human polymorphonuclear leukocyte granule proteins on Chlamydia spp. in vitro
Authors: Register K B , Davis C H , Wyrick P B , Shafer W M , Spitznagel J K
Published: January 2020

Proteins from isolated granules of human polymorphonuclear leukocytes were assessed for their nonoxidative microbicidal effect on chlamydiae by two different methods: a radioisotope assay for elementary body integrity and a biological assay for inclusion development. Crude granule extract, which consisted of a mixture of all granule proteins, caused a 20 to 30% decrease in infectivity and a 52% decrease in infectivity when incubated with Chlamydia psittaci CAL-10 and Chlamydia trachomatis serovar E, respectively. To define more specifically the components that were damaging to chlamydiae, crude granule extract was subjected to Sephadex G-75 column chromatography and isolated granule fractions were obtained. Only fractions containing lysozyme as the major component consistently caused reductions in infectivity of C. trachomatis elementary bodies. In contrast, fractions collected after the lysozyme fraction, containing proteins with molecular masses of 13,000 daltons or less, had detrimental effects on C. psittaci infectivity. Additional experiments using highly purified human polymorphonuclear leukocyte lysozyme confirmed its infectivity-reducing action upon C. trachomatis but not upon C. psittaci.
Adoptive transfer of immunity to Treponema pallidum Nichols infection in inbred strain 2 and C4D guinea pigs
Authors: Wicher V , Wicher K , Jakubowski A , Nakeeb S M
Published: January 2020

T lymphocytes purified from lymph nodes and spleens of chancre-immune, inbred strain 2 guinea pigs, when infused into syngeneic guinea pigs, conferred protection against challenge with Treponema pallidum subsp. pallidum Nichols. No protection was conferred by similar injections of cell suspensions from normal guinea pigs or guinea pigs immunized with T. phagedenis biotype Reiter or T. pallidum-free testis supernatants from infected rabbits. Similar results were obtained with homozygous C4D guinea pigs. After several months of infection, 2 of 11 strain 2 and 1 of 8 strain C4D recipients of T. pallidum-immune cells developed an erythematous reaction of short duration at the injection site; 2 of these recipients were positive for T. pallidum. Throughout the experimental period the humoral response to treponemal antigens was substantially lower in the adoptively immune guinea pigs than in various unprotected control groups. Passive immunity to infection with T. pallidum, however, seems to be dose related, since asymptomatic infection persisted for as long as 3 months after challenge in strain 2 guinea pigs transfused with 10(8) T. pallidum-immune lymphocytes, but not in C4D recipients of twice as many immune cells.
Genetic and phenotypic diversity among ampicillin-resistant, non-beta-lactamase-producing, nontypeable Haemophilus influenzae isolates
Authors: Mendelman P M , Chaffin D O , Musser J M , De Groot R , Serfass D A , Selander R K
Published: January 2020

Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-beta-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.
Demonstration of antigenic polymorphism in Plasmodium vivax malaria with a panel of 30 monoclonal antibodies
Authors: Udagama P V , David P H , Peiris J S , Ariyaratne Y G , Perera K L , Mendis K N
Published: January 2020

A panel of 30 monoclonal antibodies was established against asexual erythrocytic stages of Plasmodium vivax and used to investigate the antigenic composition of the parasite. At least 38 different antigenic polypeptides of P. vivax were characterized by the Western blot technique. The possible location of these antigens, as well as their stage and species specificity, was determined on the basis of the staining patterns produced by these antibodies on air-dried parasites in the indirect immunofluorescence test. Immunofluorescence performed with 30 different monoclonal antibodies on 50 different isolates of P. vivax obtained from patients showed that a high level of antigenic polymorphism prevailed in P. vivax. Only six monoclonal antibodies reacted with epitopes that were represented in more than 80% of parasite isolates, and therefore, appeared to be relatively conserved among different isolates. The other 24 monoclonal antibodies reacted with only 20 to 70% of parasite isolates.
Plasmid-associated resistance of Salmonella typhimurium to complement activated by the classical pathway
Authors: Vandenbosch J L , Rabert D K , Jones G W
Published: January 2020

The association of the virulence plasmid of Salmonella typhimurium with resistance to the bactericidal activity of human serum was studied in chromosomally isogenic pairs of strains differing in their virulence plasmid status. The presence of the plasmid correlated in three pairs of strains with resistance to serum. The absence of the plasmid correlated with increased sensitivity to serum, whereas the reintroduction of the plasmid to the cell resulted in the restoration of resistance to serum. Complement was activated by the classical and alternative pathways equally well by both strains of a pair, but the differential bactericidal action of serum was apparent only after the activation of complement by the classical pathway. No differences in the chemical compositions or in the molecular weight ranges of the lipopolysaccharides were apparent between paired strains. This work confirms the presence of a virulence plasmid-associated mechanism of resistance to serum and distinguishes it from lipopolysaccharide-mediated resistance.
Protective effect of human granulocyte colony-stimulating factor on microbial infection in neutropenic mice
Authors: Matsumoto M , Matsubara S , Matsuno T , Tamura M , Hattori K , Nomura H , Ono M , Yokota T
Published: January 2020

A purified human granulocyte colony-stimulating factor (hG-CSF) was studied for its protective effect on the induction of neutropenia and enhanced susceptibility to microbial infections in mice receiving cyclophosphamide (CPA). A severe reduction in peripheral blood neutrophils was induced 4 days after injection with 200 mg of CPA per kg although the level normalized rapidly thereafter. When mice were injected subcutaneously once a day with 2.5 micrograms of hG-CSF beginning on the day after CPA injection, the reduction was prevented markedly, even 4 days later. On the other hand, in mice receiving CPA 4 days prior to infection, a weakened resistance to intraperitoneal challenge with a strain of Pseudomonas aeruginosa was induced. This weakened resistance was dose-dependently restored to normal by four daily injections with hG-CSF. A daily dose of 1.0 microgram was required for complete restoration, although hG-CSF did not directly inhibit bacterial growth in vitro. In hG-CSF-treated mice, morphologically mature neutrophils migrated rapidly into the peritoneal cavities where bacteria were inoculated, followed by a rapid elimination of bacteria from the locality as compared with controls. In addition, the same treatment with hG-CSF was able to protect significantly against systemic infections caused by Serratia marcescens, Escherichia coli, Staphylococcus aureus, and Candida albicans. These data show the possibility that prophylactic therapy with hG-CSF may augment the resistance of immunocompromised patients to infections.
Introduction of Shigella flexneri 2a type and group antigen genes into oral typhoid vaccine strain Salmonella typhi Ty21a
Authors: Baron L S , Kopecko D J , Formal S B , Seid R , Guerry P , Powell C
Published: January 2020

For protection against dysentery caused by Shigella flexneri 2a, an in vivo-constructed recombinant plasmid with genes specifying the S. flexneri type and group antigens located near the pro (min 6) and his (min 44) chromosomal markers, respectively, was made and transferred to the galE Salmonella typhi strain Ty21a. Strain Ty21a carrying this recombinant plasmid was shown by immunological and biochemical analyses to express the S. flexneri 2a type and group antigens. Mice immunized with this vaccine strain were found to be protected against challenge with virulent S. flexneri 2a, but not significantly against S. typhi challenge, presumably because synthesis of the Shigella antigens interfered with expression of the typhoid antigens. Elimination of the recombinant plasmid from Ty21a allowed this strain to again express typical S. typhi O antigens. Mouse protection against both S. typhi and S. flexneri 2a challenges was achieved with a whole-cell vaccine mixture composed of equal parts of Ty21a and the Ty21a-S. flexneri 2a hybrid strain.
Antigenic characterization of the oligosaccharide portion of the lipooligosaccharide of nontypable Haemophilus influenzae
Authors: Patrick C C , Kimura A , Jackson M A , Hermanstorfer L , Hood A , McCracken G H , Hansen E J
Published: January 2020

Monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide portion of the lipooligosaccharide (LOS) of nontypable Haemophilus influenzae (NTHI) were used to characterize the LOS of this pathogen. Western blot (immunoblot) analysis with four LOS-specific MAbs and proteinase K-derived LOS preparations from 69 NTHI strains allowed the classification of these strains into nine LOS antigenic groups. The use of these MAbs in a more sensitive colony blot radioimmunoassay system together with these same NTHI strains identified 14 LOS antigenic groups. Extensive cross-reactivity was detected between the LOS epitopes of these NTHI strains and the LOS of H. influenzae type b. The epitopes recognized by these MAbs were not accessible to antibody on the surface of every strain. These LOS epitopes were also not stably expressed by NTHI growing in vitro; the observed frequency of LOS antigen variation ranged from 1 to 24% when large numbers of colonies of NTHI strains were screened for reactivity with the LOS-directed MAbs in the colony blot radioimmunoassay. This LOS antigenic variation was sometimes associated with alterations in the profile of the LOS molecule as resolved by dodecyl sulfate-polyacrylamide gradient gel electrophoresis followed by staining with silver. These data indicate that considerable antigenic diversity exists among NTHI strains with regard to the oligosaccharide epitopes in their LOS molecules.
Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters
Authors: Kim P H , Iaconis J P , Rolfe R D
Published: January 2020

In this investigation, the role of antibodies against Clostridium difficile toxins A and B in protecting hamsters against C. difficile-associated ileocecitis was examined. We also studied the transfer of protection against C. difficile-associated intestinal disease from immunized female hamsters to their infants. Adult female hamsters immunized parenterally with toxoid A or a mixture containing both toxoids A and B were protected against clindamycin-induced C. difficile-associated fatal ileocecitis. On the other hand, hamsters immunized with toxoid B or a broth filtrate from a nontoxigenic strain of C. difficile were not protected against C. difficile-induced ileocecitis. Antibody against the immunizing toxoid could be demonstrated in both the serum and the cecal contents of hamsters. Some infant hamsters from mothers immunized with toxoid A or AB were protected against C. difficile-associated ileocecitis, while infant hamsters from mothers immunized with toxoid B or a nontoxigenic broth filtrate were not protected against disease. Neutralizing antibodies to toxins A and B could be demonstrated in both maternal milk and serum, as well as in infant serum and intestinal contents. Foster-mothering experiments demonstrated that maternal protection of infants against C. difficile-associated ileocecitis was transferred to infant hamsters through breast milk. These results suggest that toxin A may play a more important role in the pathogenesis of C. difficile-associated ileocecitis in hamsters than toxin B. Furthermore, variations in the severity of C. difficile-associated illness in infants and adults may reflect the lack or presence of passively or actively acquired immunity against C. difficile toxins.
The cellular immune response to a purified antigen from Leishmania mexicana subsp. amazonensis enhances the size of the leishmanial lesion on susceptible mice
Authors: Rodrigues M M , Mendonça-Previato L , Charlab R , Barcinski M A
Published: January 2020

Immunization of BALB/c mice with gp10/20, a glycoconjugate purified from Leishmania mexicana subsp. amazonensis, induced a delayed-type hypersensitivity response to the antigen, and a significant increase was elicited in the size of the lesion induced by a subcutaneous infection with this parasite. The increase in the lesion size was observed when mice were immunized by the subcutaneous and the intraperitoneal routes. The subcutaneous immunization with gp10/20 was unable to reverse the prophylactic effect of an intravenous injection of irradiated promastigotes. An L3T4+ T-cell line specific for gp10/20 was able to transfer this lesion-enhancing effect and specific delayed-type hypersensitivity reactivity to normal syngeneic recipients. The same T-cell line was a good producer of a hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor.