bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the putative "link" glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H7 strain CL-49
Authors: Sajjan S U , Forstner J F
Published: January 2020

Purified rat intestinal mucin was used to identify mucin-binding sites for type 1-piliated Escherichia coli O157:H7 strain CL-49 isolated from a patient with hemorrhagic colitis and hemolytic uremic syndrome. Optimum binding of bacteria in a microtiter binding assay occurred with a mucin coating concentration of 15 micrograms (protein)/150 microliters. In hapten inhibition studies, several nonmucin glycoproteins bearing exposed mannosyl residues in N-linked oligosaccharides were effective inhibitors, as was rat mucin. The same glycoproteins caused bacterial aggregation. High-molecular-mass glycoproteins of the mucin were separated from its 118-kilodalton "link" glycopeptide fraction, and the latter was shown to be the mucin-binding component for E. coli CL-49 and its purified type 1 pili. This was confirmed in hemagglutination inhibition studies. Treatment of the link glycopeptide with jack bean alpha-mannosidase or endo-beta-N-acetylglucosaminidase H destroyed bacterial binding activity. Chemical or enzymatic modifications of intact rat mucin were undertaken to evaluate the normal accessibility of the link glycopeptide receptors to E. coli CL-49. Deglycosylation with trifluoromethane-sulfonic acid abolished binding, whereas pronase digestion had no effect. Reduction and alkylation as well as lipid extraction enhanced bacterial binding by the mucin, presumably by causing greater exposure of receptor sites. In summary, our binding studies revealed, for the first time, that intestinal mucin bears oligomannosyl receptors for type 1 pili and that these receptors are located on N-linked oligosaccharides of the 118-kilodalton link glycopeptide region of the mucin. Our experiments suggest the receptors are normally partly "covered" by noncovalently bound lipid. In addition, release of the link component from the rest of the mucin by disulfide bond reduction causes greater exposure of specific bacterium-binding sites.
A rns-like regulatory gene for colonization factor antigen I (CFA/I) that controls expression of CFA/I pilin
Authors: Caron J , Scott J R
Published: January 2020

Colonization factor antigens (CFA) are needed for adherence of human enterotoxigenic Escherichia coli (ETEC) strains to their hosts. The CFA/II antigens, CS1 and CS2, which are found in some ETEC strains, require the plasmid-encoded gene rns for expression (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). Other ETEC strains express CFA/I, whose synthesis and assembly require genes on two unlinked regions (regions 1 and 2) of a plasmid (G. A. Willshaw, H. R. Smith, and B. Rowe, FEMS Microbiol. Lett. 16:101-106, 1983). We report that CFA/I region 2 DNA can substitute for rns to cause expression of CS1 and CS2. The cfaR gene in region 2 is defined by a mutation abolishing both expression of CFA/I and complementation of a rns mutant for expression of CS1 or CS2. In a strain containing only region 1, complementation for expression of CFA/I by a plasmid containing rns+ is inefficient but is adequate to cause hemagglutination by the CFA/I adhesin.
Ability of mononuclear phagocytes from cattle naturally resistant or susceptible to brucellosis to control in vitro intracellular survival of Brucella abortus
Authors: Price R E , Templeton J W , Smith R , Adams L G
Published: January 2020

The role of bovine mammary macrophages and peripheral blood monocyte-derived macrophages in natural resistance to bovine brucellosis was evaluated. A group of 11 naturally resistant and 10 chronically infected susceptible cows was studied following challenge with Brucella abortus. Macrophages from a greater proportion (P less than 0.026) of naturally resistant cows were significantly superior to macrophages from susceptible cows in their ability to inhibit the in vitro intracellular replication of B. abortus after challenge exposure. Studies of a second group of cows used mammary macrophages from 12 heifers and blood monocyte-derived macrophages from 22 bulls and heifers. These tests were completed before exposure to B. abortus, using mammary macrophages (P less than 0.039) and blood monocyte-derived macrophages (P less than 0.045), and also showed that macrophages from naturally resistant cattle were significantly superior in their ability to control the in vitro intracellular replication of B. abortus. Our data indicate that the mononuclear phagocytes from more than 80% of the resistant cattle controlled intracellular replication of B. abortus significantly better than did mononuclear phagocytes from susceptible cattle. Mononuclear phagocyte function appears to be an important factor in determining natural resistance to bovine brucellosis.
Induction of interleukin-1 from murine peritoneal macrophages by Pseudomonas aeruginosa exotoxin A
Authors: Misfeldt M L , Legaard P K , Howell S E , Fornella M H , LeGrand R D
Published: January 2020

Pseudomonas exotoxin A, an ADP-ribosylating toxin produced by Pseudomonas aeruginosa, has been shown to stimulate the proliferation of murine thymocytes, which requires the participation of accessory cells. This requirement for accessory cells can be replaced by supernatant from adherent peritoneal exudate cells that have been stimulated with exotoxin A. Antibody to exotoxin A inhibits the induction of the thymocyte mitogenic activity from adherent peritoneal macrophages. However, antibody to exotoxin A had no effect on the thymocyte proliferation if the antibody was added to supernatant which contained thymocyte mitogenic activity. The thymocyte mitogenic activity was associated with a protein or protein complex with a molecular mass of greater than 10,000 daltons. D10 bioassays indicated the presence of interleukin-1 (IL-1) in the supernatant. Antibody to IL-1 inhibited the ability of supernatant to induce thymocytes to proliferate. Therefore, these data suggest that Pseudomonas exotoxin A can stimulate the production of IL-1 from adherent peritoneal cells, which induces murine thymocytes to proliferate.
Synthesis and secretion of Bordetella pertussis adenylate cyclase as a 200-kilodalton protein
Authors: Bellalou J , Ladant D , Sakamoto H
Published: January 2020

Bordetella pertussis, the etiological agent of whooping cough, synthesizes a calmodulin-sensitive adenylate cyclase that is suspected to play a major role in the virulence of this bacterium. We show that adenylate cyclase synthesized as a 200-kilodalton protein is the product of the cyaA gene and that various virulent Bordetella species secrete this high-molecular-weight polypeptide without apparent proteolytic processing. When submitted to trypsin digestion, the 200-kilodalton protein was converted to a stable 45- to 50-kilodalton species. This corresponds to the size of the enzyme previously purified from a culture supernatant. The molecular heterogeneity reported for the various identified forms of adenylate cyclase could therefore result in part from proteolytic degradation or molecular aggregation of the major 200-kilodalton form of the enzyme.
Regulation of Mycobacterium bovis BCG and foreign body granulomas in mice by the Bcg gene
Authors: Sato I Y , Kobayashi K , Kasama T , Kaga S , Kasahara K , Kanemitsu H , Nakatani K , Takahashi T , Nakamura R M , Skamene E
Published: January 2020

Natural bactericidal resistance to Mycobacterium bovis BCG is under the control of a single gene, designated Bcg. Lung granuloma formation in susceptible (Bcgs) and resistant (Bcgr) mice was studied in two sets of Bcg-congenic systems, namely, the BALB/c (Bcgs)-C.D2 (BALB/c.Bcgr) pair and the B10.A (Bcgs)-B10.Ar (Bcgr) pair, by using BCG as well as foreign body granuloma-inducing agents (dextran beads). Large granulomas of the lung induced by the intratracheal instillation of either BCG or dextran beads developed in Bcgs mice. In contrast, minimal inflammation was produced in Bcgr mice given BCG or dextran beads. Aqueous extracts prepared from pulmonary granuloma lesions induced by Bcgs mice by either BCG or dextran beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) or interleukin-4 (IL-4) activity. Very low IL-1 activity was detected in extracts from Bcgr mice injected with BCG and dextran beads. The activity of IL-1 was correlated closely with the activity and size of the granulomatous inflammation in mice. These results suggest that pleiotropic effects of the Bcg gene are involved in the development of granulomas induced by either BCG or nonspecific foreign body agents (dextran beads) and that monokines participate in granuloma formation.
Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients
Authors: Sathish M , Esser R E , Thole J E , Clark-Curtiss J E
Published: January 2020

Antigenic determinants of Mycobacterium leprae were identified by screening a lambda gt11::M. leprae genomic library with two separate pools of sera from leprosy patients. A total of 45 recombinant clones were detected with pooled sera from 21 lepromatous (LL) leprosy patients and 5 additional clones specified polypeptides that reacted with antibodies in pooled sera from 30 borderline tuberculoid or tuberculoid leprosy patients. The recombinant clones that specified antigenic determinants that reacted with sera from LL patients were condensed into eight groups on the basis of DNA hybridization experiments among the M. leprae DNA insert fragments. In addition, 11 of the 45 recombinant clones did not hybridize to members of the eight groups nor to one another; these represent unique recombinant clones. None of the recombinant clones identified by screening with sera from tuberculoid leprosy patients hybridized to each other or to any of the 45 LL recombinant clones. The polypeptides specified by the recombinant clones were usually fusion proteins with beta-galactosidase, ranging in size from 117 to 175 kilodaltons (kDa). Members of hybridization group III specified nonfusion proteins of 45 kDa. Only members of hybridization group I reacted with any of 30 monoclonal antibodies prepared against M. leprae proteins; recombinant proteins from these clones reacted with a single monoclonal antibody directed against the M. leprae 65-kDa protein. Thus, at least 22 new antigenic determinants of M. leprae have been identified on the basis of their reactivity to antibodies in sera from LL patients or sera from tuberculoid leprosy patients or both.
Use of DBA/2N mice in models of systemic candidiasis and pulmonary and systemic aspergillosis
Authors: Hector R F , Yee E , Collins M S
Published: January 2020

Mouse models of systemic candidiasis and pulmonary and systemic aspergillosis were established by using DBA/2N mice, which are known to be deficient in the C5 component of complement. In experiments comparing lethality in the respective models in DBA/2N versus outbred CFW mice, results showed that the 50% lethal dose values for the DBA/2N mice were 10- to 1,000-fold lower than those for the outbred mice, depending on the experiment. Additionally, onset of death was somewhat delayed for the DBA/2N mice. In the case of the pulmonary aspergillosis model, administration of cortisone acetate was necessary to ensure lethality after intranasal infection, but only a single dose was necessary.
Pilins from the B serogroup of Bacteroides nodosus: characterization, expression, and cross-protection
Authors: Elleman T C , Stewart D J , Finney K G , Hoyne P A , Ward C W
Published: January 2020

Sequences of pilin genes from four strains of serogroup B of the ovine pathogen Bacteroides nodosus have been determined. These sequences permit comparisons of amino acid sequence between pilins from different subtypes (B1, B2, B3, B4) of the B serogroup and assessment of intraserogroup variation. Pili of B. nodosus strains 234 (B1) and 183 (B2) were produced by Pseudomonas aeruginosa harboring a plasmid-borne B. nodosus pilin gene, and these pili were used in sheep vaccination trials. Pili from strain 183 (B2) were found to be a senior antigen to pili from strains of other B subtypes, providing protection against footrot infection caused by strains of the other B subtypes. Pili of this strain are therefore the most suitable candidate for inclusion in a pilus-based vaccine. Pili of strain 234 from subtype B1, the reference strain of the B serogroup, provided poor protection against infection with other subtypes.
Isolation and characterization of cell surface mutants of Candida albicans
Authors: Whelan W L , Delga J M , Wadsworth E , Walsh T J , Kwon-Chung K J , Calderone R , Lipke P N
Published: January 2020

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.
Signal transduction in human platelets and inflammatory mediator release induced by genetically cloned hemolysin-positive and -negative Escherichia coli strains
Authors: König B , Schönfeld W , Scheffer J , König W
Published: January 2020

Incubation of human platelets with the hemolysin-producing Escherichia coli strain K-12 (pANN5211) induced the activation of protein kinase C, aggregation of platelets, calcium influx, low amounts of 12-hydroxyeicosatetraenoic acid (12-HETE), and release of serotonin from dense granules. Nonhemolytic isogenic strains of E. coli 536/21 which differed only in their types of adhesins (MSH+ MS-Fim+; S-MRH+ S-Fim+; P-MRH+ P-Fim+) released neither serotonin nor 12-HETE from human platelets nor induced platelet aggregation. All hemolysin-negative bacteria except E. coli 536/21, without any adhesins, were able to activate protein kinase C reversibly but did not induce calcium influx. Activation of platelets with fluoride, an activator of the GTP-binding protein, was associated with protein kinase C activation, calcium influx, platelet aggregation, serotonin release, and 12-HETE formation. The simultaneous stimulation of platelets with NaF and the nonhemolytic E. coli strains suppressed several of the NaF-induced platelet responses. Membrane preparations isolated from stimulated platelets with hemolysin-negative and hemolysin-positive E. coli showed increased binding of guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and enhanced GTPase activity.
Effect of Staphylococcus aureus delta-toxin on human granulocyte functions and platelet-activating-factor metabolism
Authors: Kasimir S , Schönfeld W , Alouf J E , König W
Published: January 2020

The production of delta-toxin is supposed to be responsible for various pathophysiological effects during infection with Staphylococcus aureus. We compared the effects of delta-toxin with the structurally related bee venom toxin melittin on granulocyte functions and inflammatory mediator release. Delta-toxin and melittin induced a rapid Ca2+ influx, as was shown by fluorescence detection. Furthermore, oxygen radical production, as determined by luminol-enhanced chemiluminescence, was triggered by delta-toxin (0.15 to 15 micrograms/ml), whereas melittin showed only marginal effects. Release of lysozyme and beta-glucuronidase was observed only at high concentrations of 15 micrograms of melittin and delta-toxin per ml. Preincubation (15 min) of neutrophils with both toxins resulted in the formation of 3H-platelet-activating factor (3H-PAF) from 3H-lyso-PAF. After 5 min of incubation, the exogenously added lyso-PAF was converted to PAF (delta-toxin, 80 +/- 2%; melittin, 27 +/- 12% of total radioactivity; n = 3, mean +/- standard error of the mean) and 1-O-alkyl-2-acyl-glycerophosphorylcholine (alkyl-acyl-GPC) (corresponding values, 20 +/- 3% and 51 +/- 14% of total radioactivity). The newly generated PAF was rapidly metabolized to lyso-PAF and alkyl-acyl-GPC during the subsequent incubation period of 60 min. In the absence of any toxin, no formation of PAF from lyso-PAF was observed. Further studies indicated that the metabolism of PAF into lyso-PAF and alkyl-acyl-GPC was inhibited in the presence of delta-toxin. Melittin had no significant effects on PAF metabolism. Neither delta-toxin nor melittin modulated the uptake of PAF and lyso-PAF significantly. Our data provide evidence that delta-toxin has an effect on the activity of neutrophil granulocytes with regard to its proinflammatory capacity.
Repetitive DNA elements characteristic of pathogenic Entamoeba histolytica strains can also be detected after polymerase chain reaction in a cloned nonpathogenic strain
Authors: Mirelman D , Bracha R , Rozenblatt S , Garfinkel L I
Published: January 2020

Strains of Entamoeba histolytica which were isolated from symptomatic patients and which possess a characteristic pathogenic isoenzyme pattern (zymodeme) have extrachromosomal circular DNA molecules containing RNA genes and clusters of tandemly reiterated PvuI elements. The nucleotide sequence of comparable reiterated BamHI elements present in amebae with nonpathogenic zymodemes differs from that found in pathogenic ones. By using the polymerase chain reaction, it was demonstrated that the cloned, nonpathogenic E. histolytica strain SAW 1734R clAR also contains one or few of the tandemly repeated DNA PvuI elements characteristic of the pathogenic amebae. Sequences were detected by hybridization with the P-145 probe after in vitro amplification. Because of technical difficulties, it was impossible to resolve whether single copies of the nonpathogenic BamHI repetitive elements are present in pathogenic amebae. Our findings suggest that in the nonpathogenic amebae, the signal to start amplifying the PvuI-type elements may be induced during the process of elimination of bacterial associates from their growth environment.
Demonstration of cross-reactivity between bacterial antigens and class I human leukocyte antigens by using monoclonal antibodies to Shigella flexneri
Authors: Williams K M , Raybourne R B
Published: January 2020

Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment.
Containment as a mechanism of nonspecific enhancement of defenses against bacterial infection
Authors: Polk H C , Lamont P M , Galland R B
Published: January 2020

Observations made during the study of nonspecific enhancement of antimicrobial host defenses permitted some physiologic dissection of the anatomic basis for normal protective processes. Local growth of bacteria in the hind limb in muramyl dipeptide-treated mice was suppressed modestly, but lymphatic filtration was markedly improved, especially between the ipsilateral groin and iliac lymph nodes. Spontaneous containment, when it occurred in saline-treated controls, proceeded similarly. Certain regional lymph nodes acted to restrict the dissemination of bacteria from an anatomically isolated infection, reducing the degree of bacteremia and improving survival. Containment of local bacterial infection appears more important in a favorable host response than does dispersion and is accentuated by muramyl dipeptide.
Inhibition of Rickettsia conorii growth by recombinant tumor necrosis factor alpha: enhancement of inhibition by gamma interferon
Authors: Manor E , Sarov I
Published: January 2020

Purified human recombinant tumor necrosis factor alpha (rTNF-alpha) inhibited the growth of Rickettsia conorii (Casablanca strain) in HEp-2 cell culture. The effect was observed when the cells were pretreated with rTNF-alpha or when rTNF-alpha was added after adsorption of the rickettsiae. The inhibitory effect of rTNF-alpha on rickettsial growth was enhanced by gamma interferon. Cycloheximide had no effect on inhibition of the rickettsial yield, suggesting that de novo protein synthesis is not required for the inhibitory effect of rTNF-alpha. The addition of tryptophan partially abolished the inhibitory effect of rTNF-alpha and rTNF-alpha plus gamma interferon.
Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen
Authors: Thangaraj H S , Lamb F I , Davis E O , Jenner P J , Jeyakumar L H , Colston M J
Published: January 2020

The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences. The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme. The gene is not expressed from its own promoter in E. coli but is expressed from its own promoter in Mycobacterium smegmatis. The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined.
Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes
Authors: Boehm D F , Welch R A , Snyder I S
Published: January 2020

The calcium requirement for hemolytic activity of Escherichia coli hemolysin was investigated by using hemolytic assays and immunoblotting of toxin-treated erythrocytes. The hemolytic activity of cell culture supernatants obtained during growth of E. coli in Luria-Bertani (LB) broth or calcium-free LB broth was calcium dependent. The hemolytic activity of culture supernatants obtained during growth in LB broth supplemented with calcium was calcium independent. Osmotic protection experiments using Dextran 4 to prevent cell lysis indicated that calcium was required for the binding of hemolysin to erythrocytes at both 4 and 37 degrees C. The binding efficiency at 4 degrees C was 50% of that occurring at 37 degrees C. The calcium-dependent binding was confirmed by immunoblotting saline-washed, toxin-treated erythrocytes with a monoclonal antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. Bound hemolysin increased the calcium permeability of the cell membranes as evidenced by calcium-induced membrane protein alterations. The alterations in membrane proteins did not directly cause lysis of the cells. The results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells.
Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells
Authors: Neeper I D , Patton D L , Kuo C C
Published: January 2020

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported.
Production of toxic shock syndrome-like illness in rabbits by Staphylococcus aureus D4508: association with enterotoxin A
Authors: McCollister B D , Kreiswirth B N , Novick R P , Schlievert P M
Published: January 2020

Staphylococcus aureus D4508, obtained from a patient with nonmenstrual toxic shock syndrome (TSS), produced enterotoxin A while not making other known enterotoxins or TSS toxin 1. Concentrated culture fluids of the organism, administered subcutaneously in miniosmotic pumps, induced TSS-like symptoms (four of six animals succumbed). Identical culture fluids pretreated with anti-enterotoxin A serum failed to induce symptoms except for fever (none of six animals succumbed). Purified staphylococcal enterotoxin A also had the ability to induce TSS-like symptoms. These data suggest that enterotoxin A is the major TSS-associated toxin made by strain D4508.
Characterization of the heat shock response and identification of heat shock protein antigens of Borrelia burgdorferi
Authors: Carreiro M M , Laux D C , Nelson D R
Published: January 2020

The heat shock response of Borrelia burgdorferi B31 cells was characterized with regard to the heat shock proteins (Hsps) produced. Five to seven Hsps were detected by sodium dodecyl sulfate-gel electrophoresis and fluorography of proteins from cells labeled with [35S]methionine after shifts from 33 degrees C to 37 or 40 degrees C or from 20 degrees C to 33, 37, or 40 degrees C. Analysis of [35S]methionine-labeled Hsps by two-dimensional electrophoresis and autoradiography revealed 12 Hsps. Western immunoblot analysis with antisera to highly conserved Escherichia coli and Mycobacterium tuberculosis Hsps revealed a single 72-kilodalton (kDa) protein band that reacted with antibodies to E. coli DnaK and with antibodies to the M. tuberculosis 71-kDa Hsp homolog of E. coli DnaK. Two proteins with apparent molecular masses of 66 and 60 kDa reacted with antibodies against the M. tuberculosis 65-kDa Hsp homolog of E. coli GroEL. Human immune sera collected from patients with Lyme disease reacted with both the 66-kDa Hsp and the 60-kDa Hsp but failed to react with the 72-kDa Hsp. These data are discussed with regard to the possibility that host recognition of highly conserved epitopes of GroEL homologs of B. burgdorferi may result in autoimmune reactions causing arthritis and other pathologies.
Immunity to experimental fowl typhoid in chickens induced by a virulence plasmid-cured derivative of Salmonella gallinarum
Authors: Barrow P A
Published: January 2020

Chickens were immunized by two intramuscular inoculations at 1 and 14 days of age with virulence plasmid-cured derivatives of Salmonella gallinarum and were challenged 14 days later by oral inoculation of ca. 50 50% lethal doses (LD50) of fully virulent S. gallinarum 9. Mortality in the nonimmunized and immunized groups were 36 and 3%, respectively. This difference was highly significant (P less than 0.01). A significant reduction in mortality was also produced following oral challenge with 5,000 LD50 doses. The LD50 values by intramuscular inoculation of the challenge organism into nonimmunized and immunized chickens were log10 (0.13 +/- 1.57) and (9.74 +/- 2.72), respectively. Immunization was effective whether chickens were immunized at 1 and 14 days of age or at 21 and 35 days of age. Serum agglutinins were present in immunized chickens. Immunization with plasmid-cured Salmonella pullorum gave less protection, and immunization with Escherichia coli K-12 possessing the virulence plasmid of S. gallinarum gave none. The plasmid-cured S. gallinarum was made both rough by virulent bacteriophage activity and nalidixic acid resistant (Nalr) to produce a strain designated 9VP-phi rNalr. It was compared with a Nalr mutant of the rough 9R vaccine strain designated 9 Nalr for virulence and immunogenicity. 9VP-phi rNalr was slightly less protective and less virulent than was the 9R vaccine strain.
Dynamics of the phagocytic cell response within the lungs of parabiotic mice infected with mycobacteria with decreasing virulence for mice
Authors: Collins F M
Published: January 2020

Alveolar macrophages constitute the first line of defense against an aerogenic mycobacterial challenge. The kinetics of the alveolar macrophage response to an infectious stimulus was studied in parabiotic (C57BL/6 x DBA/2 [B6D2]F1 hybrid mice pulse-labeled with tritiated thymidine given to one (donor) animal while the other (recipient) received an equivalent amount of "cold" thymidine. Lavage fluid collected from uninfected recipients yielded few labeled monocytes. However, after introduction of 10(5) viable Mycobacterium bovis BCG into the lung, an immediate influx of heavily labeled mononuclear cells was observed, peaking around day 3. This cellular response was compared with that induced by several members of the Mycobacterium avium complex of different virulence to mice. The strains M. avium 724 and M. intracellulare 1405, virulent to mice, induced moderate mononuclear cell responses, whereas the avirulent M. intracellulare 1411 induced a predominantly polymorphonuclear rather than mononuclear cell influx, analogous to that seen when heat-killed mycobacteria were introduced into the lung. These results suggest that the mycobacteria within the lung must remain in a metabolically active state in order to induce the maximum mononuclear cell response of the type associated with acquired antituberculous immunity.
Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b
Authors: Cope L D , Yogev R , Mertsola J , Argyle J C , McCracken G H , Hansen E J
Published: January 2020

Chemical mutagenesis techniques and genetic transformation methods were used to construct isogenic mutants of Haemophilus influenzae type b (Hib) defective in the ability to synthesize lipooligosaccharide (LOS). A mutant (17B) which expressed a LOS molecule with an altered oligosaccharide was less virulent than the wild-type parent strain, as determined by measurement of the ability of these strains to produce bacteremia in infant rats after intranasal challenge. Further mutagenesis of this mutant strain yielded two new mutants with different LOS phenotypes. Mutant 7A was slightly sensitive to the bactericidal activity present in normal infant rat serum and was even less virulent than its immediate parent strain (17B) in the intranasal challenge model. However, both mutants 17B and 7A could produce bacteremia and meningitis when introduced into infant rats by the intraperitoneal route. The other LOS mutant (14A) derived from mutant 17B exhibited a level of virulence equivalent to that of the original wild-type strain. Genetic transformation of wild-type chromosomal DNA into the essentially avirulent mutant 7A and selection of transformants on the basis of their LOS antigenic characteristics resulted in the sequential restoration of full virulence to this mutant. These findings suggest that LOS is involved on at least two different levels in the ability of Hib to produce invasive disease in the infant rat model. Changes in LOS phenotype can independently affect the ability of Hib to produce bacteremia after intranasal challenge and the sensitivity of Hib to killing by normal infant rat serum. These results reinforce the significance of Hib LOS in the expression of virulence by this pathogen.
Study of epitopes of cholera enterotoxin-related enterotoxins by checkerboard immunoblotting
Authors: Kazemi M , Finkelstein R A
Published: January 2020

Checkerboard immunoblotting, a versatile new technique for examining multiple antigen and antibody interactions simultaneously, was applied in studies of epitopes in the cholera enterotoxin (CT)-related heat-labile enterotoxin (LT) family. The purified antigens used included the following: the B-subunit proteins from two CTs (CT-B-1 and CT-B-2), from classical and El Tor biotype strains of Vibrio cholerae, respectively; human LT-B-1 (H-LT-B-1) and porcine LT-B (P-LT-B) derived from LTs produced by Escherichia coli strains of human (H) and porcine (P) origins, respectively; and genetically engineered chimeric P-LT-Bs with amino acid substitutions from H-LT-B-1. The antigens were used in native, partially denatured, and CNBr-fragmented forms. The antisera included a variety of mouse monoclonal antibodies against these proteins as well as polyclonal hyperimmune sera and sera from adult American volunteer vaccinees or convalescents from induced cholera. Rabbit antisera against synthetic peptides of the CT-B-1 subunit were also used. In some instances, the effect of GM1 ganglioside on antibody binding was evaluated. The reactivity of the monoclonal antibodies was directed primarily against conformational epitopes: some were specific for homologous antigen; some were promiscuously reactive; and some recognized particular related proteins. Individual amino acids (most notably amino acid 46) exerted a dominant effect on epitope formation--in some instances, in a complementary fashion. Epitope expression was also affected by distant amino acid residues (polar effects). Some reactions were blocked by GM1 treatment of the immobilized antigen, indicating that the epitope was involved in or affected by GM1 binding. Polyclonal antibody responses varied within and among animal species. Human serum antitoxic responses were higher in convalescents from induced cholera than in recipients of a genetically engineered live vaccine, and the convalescent sera (from El Tor biotype cholera patients) generally preferred CT-B-2 to CT-B-1. The results demonstrate the potential significance of the differences among these immunologically related enterotoxins and may help provide direction to further vaccine development.
Roles of target cell membrane carbohydrate and lipid in Entamoeba histolytica interaction with mammalian cells
Authors: Bailey G B , Gilmour J R , McCoomer N E
Published: January 2020

Latex beads and liposomes carrying glycoproteins with carbohydrate sequences recognized by an Entamoeba histolytica galactose-specific binding protein were assessed for their ability to adhere to trophozoites and to stimulate amoeba actin polymerization. Glycoprotein-conjugated beads bound significantly to amoebae but did not stimulate actin polymerization. Glycoprotein-bearing liposomes bound to amoebae and did enhance actin polymerization, as do recognized glycosphingolipid-bearing liposomes (G. B. Bailey, E. D. Nudelman, D. B. Day, C. F. Harper, and J. R. Gilmour, Infect. Immun. 58:43-47, 1990). Liposome-stimulated actin polymerization occurred only if the vesicle contained negatively charged phospholipid. It was concluded that both glycoprotein and glycosphingolipid glycans on the target cell surface are involved in attachment to E. histolytica but do not themselves induce the transmembrane signals that lead to cytoskeleton activation and target destruction. This requires interaction with lipids of the target membrane bilayer.
A B cell-, T cell-linked epitope located on the adhesin of Mycoplasma pneumoniae
Authors: Jacobs E , Röck R , Dalehite L
Published: January 2020

The identification of specific T cell and B cell epitopes of the P1 protein, which functions as an adhesin and as a major antigen of Mycoplasma pneumoniae, has become of central interest for the design of synthetic vaccines. Here we report the isolation from guinea pigs infected intranasally with M. pneumoniae of hilar and bronchial T lymphocytes which proliferated after in vitro stimulation with sonicated M. pneumoniae whole-cell antigen and with the isolated P1 protein. For more detailed information on T cell epitopes, a 51-amino-acid region (histidine 821 to glycine 871; numbered from the NH2-terminal end) of the P1 protein was analyzed for a T cell epitope. An octapeptide, S-G-S-R-S-F-L-P (starting at amino acid 845), stimulated in vitro lymphocytes of bronchial washings and of hilar lymph nodes. Furthermore, this T cell-stimulating amino acid sequence was located at the C-terminal end of a B cell epitope with the sequence T-N-T (starting at amino acid 842).
Identification of a conjunctivitis-associated gene locus from the virulence plasmid of Yersinia enterocolitica
Authors: Miliotis M D , Morris J G , Cianciosi S , Wright A C , Wood P K , Robins-Browne R M
Published: January 2020

The virulence plasmid (pYV) of Yersinia enterocolitica is necessary for production of conjunctivitis in guinea pigs and for mouse lethality. To identify the genes responsible for production of conjunctivitis in guinea pigs, we subcloned the BamHI and SalI restriction fragments of the virulence plasmid of Y. enterocolitica A2635 (serotype O:8) into derivatives of the broad-host-range plasmid pRK290 and introduced the constructions into plasmid-negative Y. enterocolitica strains. A mild, transient conjunctivitis was evident 24 h after inoculation with strains containing a 2.8-kilobase (kb) BamHI fragment of pYV. These strains were cytotoxic to HEp-2 cells but did not cause death in iron-loaded adult mice. When the 2.8- and adjacent 0.5-kb BamHI fragments were deleted from the virulence plasmid of a fully virulent Y. enterocolitica isolate, the resultant strain did not cause conjunctivitis in guinea pigs and was not cytotoxic to HEp-2 cells. However, the strain with the deletion appeared to be more virulent for mice, with more rapid dissemination after orogastric inoculation, compared with that of the parent strain. When the deletion was complemented by introduction of a plasmid containing the 2.8-kb BamHI fragment, the strain again caused conjunctivitis but had decreased virulence for mice.
A new assay for invasion of HeLa 229 cells by Bordetella pertussis: effects of inhibitors, phenotypic modulation, and genetic alterations
Authors: Lee C K , Roberts A L , Finn T M , Knapp S , Mekalanos J J
Published: January 2020

Invasion and intracellular survival of Bordetella pertussis in HeLa 229 cells was studied by a new assay that utilizes polymyxin B instead of gentamicin to rapidly kill extracellular organisms. Invasion measured by this assay was time and temperature dependent and was inhibited by the microfilament drug cytochalasin D. The invasion process was also dependent on a functional vir locus (also known as bvg), the positive regulator of virulence gene expression in B. pertussis. Four spontaneous Vir- phase variants of B. pertussis and a mutant with a transposon insertion mutation in the vir locus did not invade. Cells that were environmentally modulated and thus did not express virulence determinants also did not invade. Two Vir- mutants, a vir-directed plasmid insertion mutant and a UV-light-induced mutant, were capable of invasion, although they did not produce other known virulence factors such as pertussis toxin and hemolysin but did produce small amounts of filamentous hemagglutinin (FHA) and the 69-kilodalton outer membrane protein. None of 70 Tn5 IS50L::phoA (TnphoA) insertion mutants of strain Bp18323 (including three mutants defective in FHA) tested showed any reproducible defect in invasion. A mutant carrying a site-directed deletion mutation in FHA was also capable of invasion in our assay. These data suggest that there is redundancy in the invasion functions of B. pertussis and that one or more of these are coordinately regulated with FHA and the 69-kilodalton outer membrane protein more tightly than with other vir-activated gene products.
Characterization and growth in human macrophages of Mycobacterium avium complex strains isolated from the blood of patients with acquired immunodeficiency syndrome
Authors: Meylan P R , Richman D D , Kornbluth R S
Published: January 2020

Strains of the Mycobacterium avium complex (MAC) yield opaque and transparent colonial variants when cultivated in vitro. The transparent variants are more virulent than the opaque for animals, but little is known about the respective roles of these colonial variants in humans. To assess which variant infects humans, various blood fractions from eight patients with MAC bacteremia were plated directly onto 7H10 agar. In cell fractionation studies, all the M. avium complex CFU were associated with leukocytes and none were found free in plasma. All colonies on the primary culture plate exhibited the transparent phenotype. However, during subculture in 7H9 broth or on Lowenstein-Jensen agar, opaque variants appeared in seven of eight strains. Isogenic pairs of transparent and opaque variants were prepared and used to infect in vitro human monocyte-derived macrophages from healthy seronegative individuals. Transparent variants invariably grew inside macrophages, but only one of seven opaque variants did so. These observations indicate that the bacteremia of M. avium complex in acquired immunodeficiency syndrome patients consists exclusively of the transparent variants, perhaps because these variants are able to multiply inside macrophages. In contrast, opaque variants appear after in vitro subculture and are controlled by human macrophages, consistent with their reduced virulence in animals.
Ultrastructure of phagocytosed Paracoccidioides brasiliensis in nonactivated or activated macrophages
Authors: Brummer E , Sun S H , Harrison J L , Perlman A M , Philpott D E , Stevens D A
Published: January 2020

Transmission electron microscopy was used to study ultrastructures in Paracoccidioides brasiliensis yeast cells after ingestion by nonactivated or cytokine-activated murine peritoneal macrophages. Yeast cells ingested by nonactivated macrophages had typical bi- and trilayered cell walls, plasma membranes, mitochondria, nuclei, vacuoles, etc., which remained intact for 24 h of coculture. In contrast, yeast cells ingested by activated macrophages exhibited abnormal mitochondrial ultrastructures within 4 h of interaction. Subsequent events that occurred were the formation of several clear vacuoles per cell, disintegration of the cytoplasm, and development of empty cells with intact walls. These findings provide, for the first time, insights into stepwise damage to fungal cells by activated macrophages (of particular interest in this instance because of prior evidence that the damage is due to nonoxidative mechanisms) and give possible clues regarding fungicidal mechanisms.
Adhesion of Escherichia coli strains isolated from diarrheic weaned rabbits to intestinal villi and HeLa cells
Authors: Milon A , Esslinger J , Camguilhem R
Published: January 2020

Thirty-eight strains, representative of 575 Escherichia coli isolates from weaned diarrheic rabbits, were tested for their ability to adhere in vitro to rabbit intestinal villi and to HeLa 229 cells. The O103 rhamnose-negative, highly pathogenic strains, which are epidemiologically predominant in France, attached to intestinal villi prepared from 8-day-old as well as 6-week-old rabbits and gave a diffuse adhesion pattern with HeLa cells. These adhesion properties were associated with the presence of a protein with a molecular weight of 32,000 in surface extracts of the strains. The expression of the adhesion was dependent on culture medium and temperature, and the adhesion was D-mannose resistant. Antisera raised against the 32,000-molecular-weight protein inhibited adhesion. This adhesion was not expressed in two nonpathogenic O103 strains, indicating its implication in virulence. However, the same adhesin was expressed by two O128 non- or moderately pathogenic strains. Therefore, adhesion to enterocytes is not the only factor involved in the pathogenicity of O103 strains.
Surface array proteins of Campylobacter fetus block lectin-mediated binding to type A lipopolysaccharide
Authors: Fogg G C , Yang L Y , Wang E , Blaser M J
Published: January 2020

Campylobacter fetus strains with type A lipopolysaccharide (LPS) and a surface array protein layer (S+) have been found to be pathogenic in humans and animals. Spontaneous laboratory mutants that lack surface array proteins (S-) are sensitive to the bactericidal activity of normal human serum. The ability of lectins to determine the presence of the S-layer and differentiate LPS type was assessed. We screened 14 lectins and found 3 (wheat germ agglutinin, Bandeiraea simplicifolia II, and Helix pomatia agglutinin) that agglutinated S- C. fetus strains with type A LPS but not S- strains with type B or type C LPS or S+ strains. However, the S+ type A strains were agglutinated after sequential water extraction, heat, or pronase treatment, all of which remove the S-layer, whereas there was no effect on the control strains. Specific carbohydrates for each lectin and purified LPS from a type A C. fetus strain specifically inhibited agglutination of an S- type A strain. In a direct enzyme-linked lectin assay, binding to the S- type A LPS strain was significantly greater than binding to the S+ strain (P = 0.01) or to a Campylobacter jejuni strain (P = 0.008). Consequently, these results indicate that the three lectins bind to the O side chains of C. fetus type A LPS but that the presence of the S-layer on intact cells blocks binding.