bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source
Authors: Tsai J , Dyer D W , Sparling P F
Published: January 2020

Although Neisseria meningitidis does not produce siderophores, it is able to obtain iron from human transferrin. We observed saturable specific binding of 125I-labeled human transferrin to meningococci. Human lactoferrin and mouse transferrin did not compete with human transferrin for binding, whereas human apotransferrin and 100% iron-saturated transferrin competed equally well. Meningococci thus have a specific receptor for human transferrin. Scatchard analysis yielded a relatively low Kd of 0.7 microM and an apparent copy number of 2,900 receptors per CFU. Receptor activity was iron-regulated. A meningococcal transformant specifically unable to utilize transferrin as an iron source had decreased transferrin receptor activity. These data are consistent with the hypothesis that receptor-mediated binding of transferrin is a rate-limiting step in meningococcal iron uptake from transferrin.
Proline incorporation into protein by Rickettsia prowazekii during growth in Chinese hamster ovary (CHO-K1) cells
Authors: Austin F E , Winkler H H
Published: January 2020

Both the requirement of Rickettsia prowazekii for the amino acid proline for growth and rickettsial proline incorporation were determined in Chinese hamster ovary (CHO-K1) cells auxotrophic for proline. Incubation of cells in Dulbecco modified Eagle medium supplemented with various concentrations of proline resulted in a range of host intracellular proline pools, as determined by both dansylation and equilibration of specific radioactivities. Maximal rickettsial growth was observed only in host cells with an intracellular proline pool of 1.0 mM or greater. Protein synthesis by rickettsiae in infected cells was determined to be the difference between emetine-resistant proline incorporation in the presence and absence of chloramphenicol. After density gradient centrifugation in Percoll, a rickettsial band with associated radioactivity was observed in lysates of infected cells treated with emetine but not in lysates of infected cells treated with both emetine and chloramphenicol. The average amount of proline incorporated into protein in situ was determined to be 6.3 +/- 0.8 amol per rickettsia. These results, obtained with a system which allows the study of rickettsiae in their natural habitat, are discussed in light of existing information about protein synthesis in isolated rickettsiae.
H-2-linked control of in vitro gamma interferon production in response to a 32-kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Guérin
Authors: Huygen K , Palfliet K , Jurion F , Hilgers J , ten Berg R , Van Vooren J P , De Bruyn J
Published: January 2020

A 32-kilodalton protein antigen (P32) was previously purified to homogeneity from culture filtrate of Mycobacterium bovis BCG (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Spleen cells from BCG-sensitized mice produce significant amounts of gamma interferon (IFN-gamma) in response to this P32 protein. The amount of secreted IFN-gamma is influenced by mouse genotype, with C57BL/6 (H-2b), C57BL/10 (H-2b), and 129/Sv (H-2b) mice producing about four times more than BALB/c (H-2d), CBF1 (H-2d/b), and DBA/2 (H-2d) mice do. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments indicates a probable H-2-linked control of this IFN-gamma induction, with H-2b cells producing high titers and H-2d cells producing low titers in response to the P32 antigen.
Vibrio cholerae O1 adherence to villi and lymphoid follicle epithelium: in vitro model using formalin-treated human small intestine and correlation between adherence and cell-associated hemagglutinin levels
Authors: Yamamoto T , Kamano T , Uchimura M , Iwanaga M , Yokota T
Published: January 2020

Formalin-fixed human small intestinal mucosa possessing villi and lymphoid follicle epithelium of Peyer's patches at the mucosal surface was used to test the adherence ability of clinically isolated strains of Vibrio cholerae O1. V. cholerae O1 grown on CFA agar for approximately 3 h at 37 degrees C had various levels of cellular hemagglutinins (HAs) and manifested adherence abilities that were roughly correlated with the cellular HA levels, irrespective of cellular HA types. V. cholerae O1 adhered better to epithelium over ileal lymphoid follicles than to epithelium of jejunal or ileal villi. Cells of different morphology which constituted lymphoid follicle epithelium were almost equal targets for adherence. In contrast, V. cholerae O1 grown on CFA agar for approximately 20 h at 37 degrees C in many cases had lower levels of cellular HAs and adherence abilities. Contrary to the above observations with cellular HAs and adherence, piliation of V. cholerae O1 was rather more extensive at approximately 20 h of incubation at 37 degrees C than at approximately 3 h of incubation at 37 degrees C. L-Fucose inhibited adherence to a varied extent depending on the cellular HA types, while D-mannose enhanced adherence in some strains. Heating of V. cholerae O1 diminished adherence ability. This adherence model system provides a tool by which various V. cholerae O1 strains can be preliminarily tested for adherence ability and site in human small intestine.
Analysis of the virulence of Streptococcus mutans serotype c gtfA mutants in the rat model system
Authors: Barletta R G , Michalek S M , Curtiss R
Published: January 2020

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton protein which catalyzes the synthesis of a small glucan (1.5 kilodaltons) from sucrose (J.P. Robeson, R.G. Barletta, and R. Curtiss III, J. Bacteriol. 153:211-221, 1983). To investigate the role of the GtfA enzyme in virulence, we constructed S. mutans gtfA mutants from three cariogenic serotype c strains. A plasmid that carried an erythromycin resistance determinant and an internal fragment of the gtfA gene but that was unable to replicate in streptococci was used to transform S. mutans. The erythromycin-resistant transformants carried a partial duplication of the internal gtfA fragment, because of the integration of plasmid sequences within the S. mutans gtfA gene, which also resulted in the inactivation of the gtfA gene. This was verified by Southern DNA hybridization analysis and Western blot studies of cellular protein extracts of the mutant strains with GtfA antiserum. Mutants were fully virulent in both germfree and conventional rats. These results do not rule out the involvement of the GtfA protein in virulence. Pucci and Macrina (M.J. Pucci and F.L. Macrina, Infect. Immun. 54:77-84, 1986) have suggested that the GtfA enzyme synthesizes a primer for water-insoluble glucans. Another S. mutans protein, presumably a glucosyltransferase, may have a similar function and, thus, may obscure the relevance of the GtfA enzyme in pathogenesis.
Identification and mapping of the temperature-inducible, plasmid-encoded proteins of Yersinia spp
Authors: Bölin I , Forsberg A , Norlander L , Skurnik M , Wolf-Watz H
Published: January 2020

The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. Bölin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.
Reversible phase variation of expression of Neisseria meningitidis class 5 outer membrane proteins and their relationship to gonococcal proteins II
Authors: Kawula T H , Aho E L , Barritt D S , Klapper D G , Cannon J G
Published: January 2020

Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N. meningitidis FAM18. The monoclonal antibodies also bound to class 5 proteins of a limited number of other meningococcal strains. Using the bactericidal activity of the monoclonal antibodies, we demonstrated that expression of both class 5 proteins was subject to reversible phase variation in vitro. The N-terminal amino acid sequence of a purified class 5 protein revealed striking similarity to the N-terminal amino acid sequence of gonococcal P.II proteins. Using a cloned class 5 gene, we identified three potential class 5 gene loci in N. meningitidis FAM18. These class 5 sequences also had homology with gonococcal P.II gene sequences and contained the CTCTT repeat sequence believed to be important in the regulation of gonococcal P.II expression.
Differences in excretion and efficiency of the aerobactin and enterochelin siderophores in a bovine pathogenic strain of Escherichia coli
Authors: Der Vartanian M
Published: January 2020

Secretion of aerobactin is thought to play an important part in the virulence of invasive Escherichia coli also capable of synthesizing enterochelin. Why, despite its markedly lower affinity for iron than that of enterochelin, aerobactin proves to be the predominant active siderophore for bacterial growth in transferrin was investigated. We studied the action of two iron chelators, 2,2'-dipyridyl and transferrin, in expression of the aerobactin and enterochelin genes. Specifically, we describe the sequential localization of the two siderophores in the cell compartments during bacterial growth under different iron limitation conditions. Our results demonstrated that, whatever the exogenous iron-chelating agent used, aerobactin was rapidly excreted, whereas enterochelin accumulated early in periplasm before its very belated release into the external medium. This work also showed that the advantage of aerobactin over enterochelin in competition with transferrin was not due to (i) lack of enterochelin activity, (ii) a cell-bound aerobactin-dependent mechanism, (iii) antagonism between the two siderophores, and probably (iv) genetic preferential induction of aerobactin. We propose that the superiority of aerobactin in competing with transferrin for iron(III) was a consequence of its more rapid excretion with respect to enterochelin. In contrast to transferrin, 2,2'-dipyridyl induced a greater efficiency of enterochelin, possibly by a more permanent function as iron-binding compounds in the bacterial envelope. In summary, unlike aerobactin, enterochelin appears to be a weakly secreted high-affinity iron ligand.
The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein
Authors: Shinnick T M , Vodkin M H , Williams J C
Published: January 2020

Monoclonal hybridoma antibodies directed against a 65-kilodalton (kDa) mycobacterial protein could detect similarly sized antigens in many other bacterial species. In Pseudomonas aeruginosa, the cross-reacting protein corresponded to a 62-kDa antigen that has been called Common Antigen. The mycobacterial 65-kDa antigen and Common Antigen are similar in that both (i) are highly immunoreactive molecules, (ii) contain species-specific and genus-specific epitopes in addition to the broadly cross-reactive epitopes, (iii) can be isolated as homomultimers of greater than 240 kDa, and (iv) have similar amino acid compositions. In Escherichia coli, the cross-reactive protein corresponded to the GroEL protein. Both the GroEL protein and the mycobacterial 65-kDa protein are expressed as heat shock proteins.
Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae
Authors: Dudas K C , Apicella M A
Published: January 2020

The identification of enterobacterial mutants that contain alterations in the lipopolysaccharide (LPS) oligosaccharide core structure facilitated the development of the model of the physicochemical and immunochemical structures of enteric LPS. Results of recent immunochemical studies have suggested that the structural model of the lipooligosaccharides (LOSs) of Neisseria gonorrhoeae may differ from the enteric LPS model. The difficulties in the analysis of the wild-type gonococcal LOS have precluded understanding of the precise nature of the LOS structure. This study was undertaken to isolate a series of mutants of N. gonorrhoeae 1291 that had sequential saccharide deletions in the LOS. Results of preliminary studies suggested that the pyocin, designated pyocin C, allowed selection of gonococci with such mutant LOS structures. Results also indicated that the receptor for pyocin C binding was an LOS component. Pyocin C selection led to the isolation of five strains with LOS patterns on sodium dodecyl sulfate-polyacrylamide gels which differed from the LOS of parent strain 1291. In this system, the Mr of the parent LOS was 4,715, while the LOSs from the mutant strains demonstrated progressive saccharide deletions, with Mrs of 4,230, 4,089, 3,627, 3,262, and 3,197. Protein patterns of these mutants on sodium dodecyl sulfate-polyacrylamide gels were qualitatively similar to those of the parent strains. Results of studies with five monoclonal antibodies specific for neisserial LOS indicated that shared as well as unique epitopes were present on the mutant LOSs. Results of ketodeoxyoctonate analysis of the mutant LOSs indicated that the majority of the ketodeoxyoctonate residues may be substituted on C-4 or C-5. Chemical and immunological analysis of such LOS mutants should expedite the development of the model for the structure of gonococcal LOS.
Attaching and effacing adherence of Vero cytotoxin-producing Escherichia coli to rabbit intestinal epithelium in vivo
Authors: Sherman P , Soni R , Karmali M
Published: January 2020

Vero cytotoxin-producing Escherichia coli (VTEC) strains have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. Since adherence of enteric pathogens to epithelial surfaces is often a prerequisite for the subsequent delivery of bacterial enterotoxins and mucosal invasion, we evaluated intestinal adherence by 18 VTEC strains, which were of human origin and belonged to 10 distinct serotypes, 7 days after enteral challenge of rabbits. A total of 23 postweaning rabbits (body weight, 1 kg) were each fed 2 X 10(8) VTEC, and 5 rabbits were challenged with an equal number of fecal commensal E. coli strains as controls. Each rabbit was monitored daily for the development of diarrhea. At 7 days after infection the proximal jejunum, distal ileum, cecum, and proximal colon were removed from each rabbit and examined for the presence of adherent organisms under light microscopy, after Giemsa staining of Formalin-fixed secretions, and by transmission electron microscopy. Nonbloody diarrhea developed in 16 of 23 VTEC-infected rabbits in contrast to 0 of 5 infected with commensal E. coli strains (P less than 0.02). Organisms were adherent to surface epithelial cells in the ceca (20 of 23 rabbits), proximal colons (9 of 23), and distal ilea (6 of 23) of VTEC-infected rabbits. Intimate attaching and effacing binding of bacteria to intestinal epithelial cells, in regions where the normal microvillous membrane architecture had been disrupted, was observed under electron microscopy for VTEC of multiple serotypes. In contrast, no organisms were adherent to the jejuni. Adherence of organisms was not seen in any portion of the intestines of rabbits that were challenged with commensals. These findings indicate that multiple serotypes of VTEC demonstrate intimate attaching and effacing binding to rabbit enterocytes and colonocytes in vivo. In addition to bacterial binding in the ceca and colons, VTEC adhere to enterocytes in the distal small intestines of per orally infected postweaning rabbits.
Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953
Authors: Takada H , Ogawa T , Yoshimura F , Otsuka K , Kokeguchi S , Kato K , Umemoto T , Kotani S
Published: January 2020

From Fusobacterium nucleatum ATCC 10953 cell envelope fraction whose inner membranes had been removed by treatment with sodium N-lauroyl sarcosinate, an outer membrane protein (37,000 Mr in a native state) was prepared by extraction with lithium dodecyl sulfate. The protein thus obtained showed distinct porin activity, namely, the ability to form hydrophilic diffusion pores by incorporation into the artificial liposome membrane. The porin fraction exhibited strong immunobiological activities in the in vitro assays: B-cell mitogenicity and polyclonal B-cell activation on murine splenocytes, stimulatory effects on guinea pig peritoneal macrophages, and enhancement of the migration of human blood monocytes. The porin fraction also exhibited immunoadjuvant activity to increase the antibody production against sheep erythrocytes in the spleen of mice that were immunized by sheep erythrocytes with porin. Although chemical analyses revealed that the test porin fraction contained a considerable amount of lipopolysaccharide (LPS) (around 12% of the fraction), the studies with LPS-nonresponding C3H/HeJ mice and on the inhibitory effects of polymyxin B strongly suggest that most of the above bioactivities are due to porin protein itself, not to coexistent LPS in the porin fraction.
Establishment of a monoclonal antibody recognizing an antigenic site common to Clostridium botulinum type B, C1, D, and E toxins and tetanus toxin
Authors: Tsuzuki K , Yokosawa N , Syuto B , Ohishi I , Fujii N , Kimura K , Oguma K
Published: January 2020

The partial amino acid sequence of the light-chain (Lc) component of Clostridium botulinum type C1 toxin was determined. The sequence was quite similar to those of the other types of botulinum and tetanus toxins. Nine monoclonal antibodies against botulinum type E toxin were established by immunizing BALB/c mice with type E toxoid or its Lc component. Six antibodies reacted with the heavy-chain component and three reacted with the Lc component of the toxin. One of the latter three antibodies reacted with botulinum type B, C1, and D toxins and tetanus toxin, as well as botulinum type E toxin. This antibody recognized the Lc components of these toxins, indicating that there exists one common antigenic determinant on the Lc regions of these toxins.
Combined parenteral and oral immunization results in an enhanced mucosal immunoglobulin A response to Shigella flexneri
Authors: Keren D F , McDonald R A , Carey J L
Published: January 2020

Achieving a vigorous secretory immunoglobulin A (IgA) response in intestinal secretions usually requires multiple doses of antigen given orally, while systemic immunity is more easily attained by parenteral immunization. This study examines the role of combined parenteral and oral immunizations to enhance the early mucosal immune response to an enteropathogen. We have used a chronically isolated intestinal-loop model in rabbits as a probe to monitor kinetically the initial (primary) local immune response to shigella lipopolysaccharide (LPS) following combinations of parenteral immunization intramuscularly (i.m.) and oral stimulation with shigellae. Predictably, effective stimulation of systemic immunity was elicited when heat-killed preparations of Shigella sp. strain X16 were given i.m., as shown by strong serum IgG and weak intestinal IgA activity to shigella LPS. A single oral dose of live Shigella sp. strain X16 given to unprimed rabbits elicited only a typical weak IgA response in intestinal secretions. However, when an i.m. dose of heat-killed shigellae was followed 1 day later by an oral dose of live Shigella sp. strain X16, a hyperstimulation of the early secretory IgA response was elicited, and the response reached levels found previously only after multiple oral administrations of live shigellae. This stimulation did not require the use of an adjuvant. At the same time, the animals receiving this combined oral and i.m. regimen had a lower IgG antishigella LPS activity in serum compared with their response after receiving parenteral antigen in adjuvant alone. These findings indicate that while a dichotomy exists between the systemic and mucosal immune responses, careful orchestration of the stimulatory events can promote a vigorous early local IgA response.
Outer membrane protein F preparation of Pseudomonas aeruginosa as a vaccine against chronic pulmonary infection with heterologous immunotype strains in a rat model
Authors: Gilleland H E , Gilleland L B , Matthews-Greer J M
Published: January 2020

Outer membrane protein F (porin) was purified by extraction from polyacrylamide gels of cell envelope proteins of the Pseudomonas aeruginosa PAO1 strain. Rats were immunized intramuscularly with 25 micrograms of protein F on days 1 and 14 and then challenged on day 28 via intratracheal inoculation of bacterium-containing agar beads. On day 35 the lungs were either fixed for histological examination or submitted for quantitation of the bacteria present. protein F immunization afforded significant protection against challenge with six of six heterologous lipopolysaccharide immunotype strains of P. aeruginosa. By an enzyme-linked immunosorbent assay, the protein F-immunized rats had both immunoglobulin G and M antibody responses to cell envelopes of all six of the heterologous immunotype strains. Protein F immunization greatly enhanced the ability of the rats to clear the inoculated P. aeruginosa from the lungs and significantly reduced the incidence and severity of pulmonary lesions as compared with those in bovine serum albumin-immunized control rats. These data show the efficacy of outer membrane protein F as a protective vaccine in a rat model of chronic pulmonary infection.
Modified lymphocyte response to mitogens after intraperitoneal injection of glycopeptidolipid antigens from Mycobacterium avium complex
Authors: Brownback P E , Barrow W W
Published: January 2020

Intraperitoneal injection of glycopeptidolipid (GPL) antigens from Mycobacterium avium complex serovar 4 resulted in the decreased ability of murine splenic lymphocytes to respond to nonspecific-mitogen-induced blastogenesis when exposed to concanavalin A, phytohemagglutinin, and lipopolysaccharide. Adherent cell depletion and cell mixing experiments with T lymphocytes indicated that macrophages were not a major contributor to the immunosuppression observed in this study. Enumeration of splenic lymphocytes by means of flow-cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies demonstrated that intraperitoneal injection of GPL antigens resulted in a significant decrease in Thy-1+ and Lyt-1+ cells but no change in the numbers of Lyt-2+ cells. Treatment with GPL antigens in vitro affected the ability of splenic mononuclear cells to respond optimally for concanavalin A-induced blastogenesis at 40 micrograms of GPL per 4 X 10(5) cells per 0.2 ml and lipopolysaccharide-induced blastogenesis at concentrations ranging from 5 to 40 micrograms of GPL per 4 X 10(5) cells per 0.2 ml. However, in vitro treatment with GPL antigens did not affect phytohemagglutinin-induced blastogenesis at concentrations ranging from 5 to 40 micrograms of GPL per 4 X 10(5) cells per 0.2 ml. These findings suggest that GPL antigens or their metabolites affect lymphocyte function and may be important cofactors in the overall pathogenesis of M. avium complex infections.
Purification and analysis with monoclonal antibodies of P2, the major outer membrane protein of nontypable Haemophilus influenzae
Authors: Murphy T F , Bartos L C
Published: January 2020

The protein P2 comprises a large proportion of the outer membrane of nontypable Haemophilus influenzae and functions as a porin. In view of the importance of the protein as a surface antigen, the present study was designed to purify and analyze P2 with particular emphasis on detection of antigenic determinants expressed on the bacterial surface and identification of bactericidal targets on P2. The P2 protein was purified by using detergent solubility, anion-exchange chromatography, and gel-filtration chromatography sequentially. Two monoclonal antibodies to P2 were developed. One antibody (2E6) recognized a determinant expressed on the bacterial surface, whereas the other antibody (3F3) recognized an internal epitope. The surface-exposed 2E6 determinant was present on 12% of strains from a nationwide collection. P2 is a bactericidal target for antibody 2E6. Cyanogen bromide cleavage of P2 resulted in two fragments, as in type b strains. Both monoclonal antibodies recognized epitopes on the larger fragment. These observations have potentially important implications regarding the development of vaccines to prevent H. influenzae infections and the development of a serotyping system for epidemiologic studies.
Characterization of Brucella polysaccharide B
Authors: Bundle D R , Cherwonogrodzky J W , Perry M B
Published: January 2020

Polysaccharide B was extracted from Brucella melitensis 16M and from a rough strain of Brucella abortus 45/20 by autoclaving or trichloroacetic acid extraction of whole cells and by a new method involving mild leaching of cells. The material obtained by either of the established procedures was contaminated by O polysaccharide. The new leaching protocol eliminated this impurity and provided a pure glucan, which was regarded as polysaccharide B. This polysaccharide was found by high-performance liquid chromatography separations, chemical composition, methylation, and two-dimensional homo- and heteronuclear magnetic resonance experiments to be a family of nonreducing cyclic 1,2-linked polymers of beta-D-glucopyranosyl residues. The degree of polymerization varied between 17 and 24. Polysaccharide B was essentially identical to cyclic D-glucans produced by Rhizobia, Agrobacteria, and other bacterial species. Pure polysaccharide B did not precipitate with Brucella anti-A or anti-M serum and did not inhibit the serological reaction of Brucella A or M antigen with either bovine or murine monoclonal Brucella anti-A or anti-M serum. Previously described serological reactions of polysaccharide B preparations with Brucella anti-A and anti-M sera are related in this study to the presence in crude extracts of contaminants with the antigenic properties of Brucella lipopolysaccharide O polysaccharides.
Clostridium difficile toxins A and B inhibit human immune response in vitro
Authors: Däubener W , Leiser E , von Eichel-Streiber C , Hadding U
Published: January 2020

Two Clostridium difficile toxins isolated from strain VPI 10463 were tested for their effect on different human T-cell proliferation systems. In mitogen- and antigen-driven T-cell proliferation systems, toxins inhibited the proliferative response in a dose-dependent fashion. In interleukin-2-driven culture systems, no effect of toxins could be found on preactivated T cells. We suspected that monocytes were the influenced cells, since in antigen- and mitogen-driven systems monocytes were necessary for the proliferative response, whereas the interleukin-2-driven system was independent of monocytes. To prove this concept, purified monocytes were treated with toxins. The treatment was found to markedly reduce the capacity of monocytes to stimulate T-cell proliferation. No inhibition of the proliferative response was measured when, instead of monocytes, resting or preactivated T cells were treated with toxins. These experiments clearly show that C. difficile toxins interact with monocytes and not with T cells. The effect of toxins on cells of the immune system might be one factor in the development of pseudomembranous colitis.
Cellular defense of the avian respiratory system: influx and nonopsonic phagocytosis by respiratory phagocytes activated by Pasteurella multocida
Authors: Toth T E , Pyle R H , Caceci T , Siegel P B , Ochs D
Published: January 2020

Poultry have a very limited number of resident macrophages in the normal steady-state respiratory tract. Thus, poultry must rely heavily on active migration of phagocytic cells to the lungs and air sacs in defending against respiratory pathogens. Intratracheal administration of a live, apathogenic Pasteurella multocida vaccine (Choloral; Clemson University strain) increased the number of avian respiratory phagocytes (ARP; obtained by lavage of lungs and air sacs) within 24 h by 3 orders of magnitude compared with the number of ARP obtained from mock-inoculated controls and from nonreacting chickens. Chickens yielding a high number of ARP did not show any sign of respiratory disease. Flow cytometric analysis of ARP that were exposed to 20 nonopsonized fluorescent microspheres per ARP for 30 min at 37 degrees C demonstrated a fivefold increase in the percentage of actively phagocytic cells in the ARP populations of stimulated chickens compared with the percentage of phagocytic ARP for mock-inoculated control birds. The phagocytic capacity (relative number of engulfed microspheres) of ARP from stimulated birds doubled during the same time. The flow cytometric observations were confirmed by fluorescence microscopy. These results indicate that activation by avirulent replicating agents of phagocytic cells of chicken to migrate to the respiratory tract may be a means of defending poultry against air sacculitis and pneumonia.
A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes
Authors: Sandström G , Löfgren S , Tärnvik A
Published: January 2020

The live vaccine strain (LVS) of Francisella tularensis is killed by human polymorphonuclear leukocytes as a result of strictly oxygen-dependent mechanisms (S. Löfgren, A. Tärnvik, M. Thore, and J. Carlsson, Infect. Immun. 43:730-734, 1984). We now report that a capsule-deficient (Cap-) mutant of LVS survives in the leukocytes. In contrast to the encapsulated parent strain, the Cap- mutant was avirulent in mice and was susceptible to the bactericidal effect of nonimmune human serum. The mutant was killed by serum as a result of activation of the classical pathway of complement by naturally occurring immunoglobulin M. This killing by serum was mitigated by the presence of human polymorphonuclear leukocytes. After opsonization in complement component C5-deficient nonimmune serum, the Cap- mutant was ingested and survived in the leukocytes. Under these conditions, the parent strain was killed. The leukocytes responded to both the parent and the Cap- strain with a very low chemiluminescent response. Only the response to the parent strain was inhibited by superoxide dismutase. When the Cap- mutant was opsonized with immunoglobulin G, it induced a higher and superoxide dismutase-inhibitable chemiluminescent response and was killed by the leukocytes. In conclusion, the capsule of F. tularensis LVS seemed to protect this organism against the bactericidal effect of serum. When deprived of the capsule, the organism failed to induce an antimicrobial response in polymorphonuclear leukocytes and survived in the leukocytes. Survival in phagocytes is a key characteristic of intracellular parasites. The Cap- mutant of F. tularensis may become a useful tool in experiments to explain the differences between pathways of ingestion of intracellular parasites, evidenced by the death or survival of the parasite.
Purification of serotype I antigen from nutritionally variant streptococci
Authors: George M , van de Rijn I
Published: January 2020

The nutritionally variant streptococci (NVS) are a fastidious group of bacteria first recognized in the early 1960s in the blood cultures of patients with subacute bacterial endocarditis. Since that time, the NVS have been implicated in 5 to 6% of all cases of human bacterial endocarditis. The NVS possess membrane-associated amphipathic molecules different from those described for other streptococci. Unitl recently, chemical characterization of these new amphipathic polymers was hampered by unsuccessful attempts at isolating large quantities of these molecules in a form free from other bacterial components. Presently, stationary-phase-culture supernatants provide an optimum source of crude material for amphiphile purification procedures. Hydrophobic-interaction chromatography in conjunction with immunoaffinity chromatography yields an NVS serotype I amphiphile preparation free of contaminants, as determined by immunoelectrophoretic and chemical analyses. Tandem crossed immunoelectrophoresis of the purified extracellular NVS amphiphile demonstrated that it is immunologically similar to the intracellular amphiphile. Finally, this amphiphile serves as the NVS serotype I antigen.
Characterization of P fimbriae on O1, O7, O75, rough, and nontypable strains of Escherichia coli
Authors: Pere A , Selander R K , Korhonen T K
Published: January 2020

P fimbriae of 37 uropathogenic Escherichia coli O1:K1, O7:K1, O22, O75, rough:K1, and nontypable strains were characterized by immunoprecipitation with 14 fimbria-specific rabbit antisera. The fimbrial composition of these strains, as reflected by the apparent molecular weights of the fimbrial peptides, was correlated with the O serogroup of the strains, but serological cross-reactivity of P fimbriae of different E. coli serogroups was frequently observed. The genetic clonal relationships of the strains were analyzed by determining the electrophoretic types, based on 18 chromosomally encoded enzymes. Among the O1:K1 strains, the same P-fimbrial variants occurred on strains that were either closely related or very distinct in their electrophoretic types, indicating that the P fimbriae have evolved in association with the O and K antigens. In contrast, certain O7:K1 and R:K1 strains as well as some O22 and O75 strains were genotypically identical and shared similar P-fimbrial variants, which differed serologically from those of other E. coli serogroups. Our results show that, despite the structural variability seen in electrophoretic analysis of P fimbriae of different serogroups, many P-fimbrial variants share common antigenic determinants that are recognized by rabbit antisera. Based on immunoprecipitation analyses, three anti-P-fimbria sera have now been identified that react with P fimbriae of 82 of 84 uropathogenic E. coli strains characterized in Finland.
Protective antigens against enterotoxigenic Escherichia coli O101:K99,F41 in the infant mouse diarrhea model
Authors: Duchet-Suchaux M
Published: January 2020

Protective antigens present in whole cells of bovine enterotoxigenic Escherichia coli strains were tested in an infant mouse diarrhea model. Reduction of mortality rates of infant mice suckling vaccinated mothers was measured 1, 2, and 5 days after oral challenge with strain B41 (O101:K99,F41:H-). Vaccines consisted of strains of serogroup O101, O9, O8, or O20 and of variants bearing or not bearing antigens K99 and F41. K99 and F41 expressions were checked by slide agglutination with K99 and F41 antisera and by mannose-resistant microhemagglutination with horse, sheep, and guinea pig erythrocytes. Absence of production of K99 antibodies following hyperimmunization with K99-negative variants was established in rabbit antisera. Strains bearing K99 alone induced less protection of infant mice 5 days after challenge than strains bearing F41 alone or both K99 and F41. Absence of expression of K99 in the vaccinal strains resulted in either a slight decrease (strains bearing additional F41) or complete abolishment (strains bearing K99 alone) of protection. Failure of F41 synthesis by O101 strains or variants resulted in no protection 5 days after challenge. F41 probably also supplied most of the protection induced by the O9 strain. When negative for both K99 and F41, strains of serogroup O101 still provided protection 1 and 2 days after challenge. This protection was also induced by strain H510a, the reference for O101 antigen. Thus, O antigen contributed to the best vaccinal protection, in addition to K99 and F41.
Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes
Authors: Mintz C S , Chen J X , Shuman H A
Published: January 2020

Attempts to isolate auxotrophic mutants of Legionella pneumophila have been hampered by the complex nutritional composition of the media used to cultivate this organism. We developed a semidefined medium, designated CAA, to facilitate the isolation and characterization of Legionella auxotrophs. Unlike previously described chemically defined media for this organism, L. pneumophila formed colonies on CAA agar. Using this medium, we isolated several independent tryptophan auxotrophs of strain Philadelphia-1 after ethyl methanesulfonate mutagenesis and penicillin enrichment. Trimethoprim selection was used to isolate several independent thymidine-requiring mutants of the same strain. The thymidine auxotrophs exhibited a marked decrease in viability when they were deprived of thymidine. The results of monocyte infection experiments with both the tryptophan and thymidine auxotrophs indicated that the thymidine auxotrophs were incapable of intracellular survival or multiplication. In contrast, the tryptophan auxotrophs grew well in monocyte cultures. The isolation of additional auxotrophic mutants will facilitate the study of the nutritional requirements of L. pneumophila for growth in human mononuclear phagocytes.
Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica
Authors: Weikel C S , Murphy C F , Orozco E , Ravdin J I
Published: January 2020

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.
Molecular analysis of enterotoxin plasmids of enterotoxigenic Escherichia coli of 14 different O serotypes
Authors: Danbara H , Komase K , Arita H , Abe H , Yoshikawa M
Published: January 2020

A total of 104 isolates of enterotoxigenic Escherichia coli derived from diarrheal patients from more than 10 countries were examined for serotype and toxigenicity. The transferability and molecular structure of the enterotoxin plasmids from each isolate were also examined. Enterotoxin plasmids from serotypes such as O6, O25, O27, O126, O128, and O159, which are frequently associated with E. coli diarrhea (classical strains) generally did not transfer by conjugation from clinical isolates, whereas those from serotypes such as O7, O17, O80, O98, O139, O150, and O153, which are rarely associated with diarrhea (rare strains) transferred almost always from the clinical isolates by conjugation. Analyses of enterotoxin plasmids by restriction endonucleases and DNA-DNA hybridization with the enterotoxin probes revealed that the strains with the same O serotype and toxigenicity carry closely related enterotoxin plasmids. These results suggest that classical strains resulted from the dissemination of ancestral clones which received enterotoxin plasmids long ago, while the rare strains acquired the enterotoxin plasmids recently by conjugation and have not yet been spread to the same degree as the ancestral clones.
Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits
Authors: Lu Y S , Afendis S J , Pakes S P
Published: January 2020

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.
Sequence analysis of the glucosyltransferase A gene (gtfA) from Streptococcus mutans Ingbritt
Authors: Ferretti J J , Huang T T , Russell R R
Published: January 2020

The complete nucleotide sequence of a 2.4-kilobase fragment containing the glucosyltransferase A gene (gtfA) of Streptococcus mutans Ingbritt (serotype c) was determined. The gtfA gene contains 481 codons and specifies a protein of molecular weight 55,665. There is no evidence of a signal peptide in the protein or that the glucosyltransferase A enzyme is secreted. No sequence homologies were observed between the gtfA gene or protein and the gtfI or gtfB gene and its protein.
Proteins that appear to be associated with pili in Neisseria gonorrhoeae
Authors: Muir L L , Strugnell R A , Davies J K
Published: January 2020

Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated.
Characterization of the plasmid from Escherichia coli RDEC-1 that mediates expression of adhesin AF/R1 and evidence that AF/R1 pili promote but are not essential for enteropathogenic disease
Authors: Wolf M K , Andrews G P , Fritz D L , Sjogren R W , Boedeker E C
Published: January 2020

RDEC-1, an Escherichia coli strain that adheres to rabbit mucosa and causes an attaching, effacing lesion, expresses the pilus adhesin AF/R1 which determines in vitro attachment to rabbit intestinal brush borders. In order to determine the role of AF/R1 pili in the pathogenesis of enteropathogenic diarrhea in rabbits, we localized the genes for AF/R1 expression, constructed an AF/R1- strain, and compared the virulence of the AF/R1+ and AF/R1- strains with particular attention to the development of attaching, effacing lesions. We introduced Tn5 into the 86-megadalton (MDa) conjugative plasmid known to mediate expression of AF/R1 pili and transferred the derivative plasmids into laboratory strain HB101. Transconjugant M5 was found to contain the 86-MDa plasmid from RDEC-1 and to express AF/R1 pili. Pilus expression on M5 was confirmed by reaction with antiserum raised against purified AF/R1 pili and allowed the bacteria to adhere to the rabbit ileum in an in vitro assay. Three Tn5 insertions in the 86-MDa plasmid were obtained which resulted in loss of AF/R1 expression. Part of the plasmid was mapped, including a region necessary for AF/R1 pilus expression. AF/R1- mutant strain M34 was constructed, and its pathogenesis was investigated. M34 produced disease in rabbits but was less virulent than the parent. The characteristic effacing lesions of RDEC-1 and enteropathogenic E. coli developed in the intestine of rabbits infected with either M34 or RDEC-1, although with M34 they were much less frequent and did not involve the small bowel. We conclude that AF/R1 pilus expression is not essential for the attaching, effacing lesion but serves as an accessory virulence factor which promotes an initial interaction of RDEC-1 with normal epithelial cells.
Oxidative requirement for degranulation of human peripheral blood eosinophils
Authors: Baskar P , Pincus S H
Published: January 2020

Eosinophils possess both oxygen-dependent and oxygen-independent mechanisms for damaging helminthic parasites such as schistosomula. We have studied the release of the granular enzymes beta-glucuronidase and arylsulfatase to evaluate the oxidative requirement for degranulation. Both ionophore-mediated and immunoglobulin G-mediated release of granular enzymes were enhanced in the presence of oxygen (P less than or equal to 0.05). Calcium ionophore-mediated degranulation under aerobic conditions was reduced by the addition of the degradative enzymes catalase and superoxide dismutase, suggesting that active oxygen products enhance degranulation. In contrast, oxygen products did not appear to contribute to degranulation induced by immunoglobulin G-coated beads.