bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein
Authors: Goroncy-Bermes P , Dale J B , Beachey E H , Opferkuch W
Published: January 2020

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.
In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2
Authors: Gaillard J L , Berche P , Mounier J , Richard S , Sansonetti P
Published: January 2020

Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.
Allergenic and blastogenic reactivity of three antigens from Mycobacterium tuberculosis in sensitized guinea pigs
Authors: Worsaae A , Ljungqvist L , Hasløv K , Heron I , Bennedsen J
Published: January 2020

Three antigens from a culture filtrate of Mycobacterium tuberculosis H37Rv were purified by affinity chromatography, using monoclonal antibodies. The molecular weights of the purified antigens are 17,000 to 19,000, 32,000 to 33,000, and 39,000, respectively, and by their migration patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, they all appeared to be single-chain polypeptides. Western blot and enzyme-linked immunosorbent assay analyses indicated that the antigens are non-cross-reactive. All antigens generated an intermediate to strong skin reaction when tested in guinea pigs previously immunized with a live M. bovis BCG vaccine or with an oil emulsion preparation of phenol-or heat-killed M. tuberculosis. Lymphocytes isolated from peripheral blood or lymph nodes of similarly immunized guinea pigs could be stimulated by purified protein derivative and the purified antigens. Qualitative differences in stimulatory capacity between the preparations were demonstrated. The antigens may prove useful in further studies of the immunology and pathogenesis of tuberculosis.
Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody
Authors: Hugo F , Sinner A , Reichwein J , Bhakdi S
Published: January 2020

A murine monoclonal antibody generated against staphylococcal alpha-toxin was shown to react only with the monomeric (native), 3S form of the toxin. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) constructed with this antibody permitted detection of 0.25 to 0.5 ng of native toxin per ml. Toxin oligomers formed either by heat aggregation in solution, on target erythrocyte membranes, or on phosphatidylcholine-cholesterol liposomes were unreactive in the ELISA when membranes were solubilized with the nondenaturing detergent Triton X-100. After dissociation of the oligomers by boiling in sodium dodecyl sulfate, however, the ELISA reactivity of the liberated 3S toxin was fully restored. Parallel determinations of membrane-bound toxin with sodium dodecyl sulfate and Triton X-100 solubilization thus permitted direct quantitation of total and monomeric toxin, respectively; the difference between these two values was represented by toxin oligomers. The detection limits for membrane-bound oligomeric and monomeric toxin on erythrocyte membranes are in the order of 100 molecules and 1 molecule per cell, respectively. Using this ELISA, we show that over 90% of alpha-toxin molecules bound to target membranes at 37 degrees C are in oligomeric form. Evidence is given that the monoclonal antibody neutralizes alpha-toxin by inhibiting its binding to both rabbit and human erythrocytes. This ELISA is the first assay that quantitatively discriminates between mono- and oligomeric forms of a pore-forming protein on target cell membranes.
Transfer of resistance to primary infection of Listeria monocytogenes and early induction of delayed hypersensitivity by sera from L. monocytogenes-infected mice
Authors: Yamada A , Himeno K , Nakamura S , Kawamura I , Nomoto K
Published: January 2020

We found a new phenomenon which differs from previous reports on experimental listeriosis, that is, failure of passive transfer of serum from Listeria monocytogenes-infected mice to convey resistance to the bacterium. Transfer of immune serum from L. monocytogenes-infected mice markedly augmented resistance to the bacterium, and mechanisms of the transfer of L. monocytogenes-immune serum were investigated. Transfer of immune serum prevented L. monocytogenes lethality. This effect of the immune serum was transferred dose dependently. Augmentation of resistance to L. monocytogenes also appeared in elimination of bacteria from the spleen. The growth of bacteria within 2 days in the spleen was not inhibited. Transfer of the immune serum augmented and accelerated induction of a delayed footpad reaction. Delayed hypersensitivity-dependent accumulation of mononuclear cells, detected by focus formation reaction in the liver, was also augmented. In contrast, polymorphonuclear cell accumulation in the liver was suppressed. Development of delayed hypersensitivity reactions was correlated with the elimination of bacteria in the spleens. These effects of the immune serum were expressed antigen specifically; however, the effector molecule(s) in the immune serum differs from immunoglobulin molecules.
Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages
Authors: James S L , Glaven J A
Published: January 2020

Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target.
Absence of significant cellulase activity in microbial flora of the female genital tract
Authors: Garland S M , Tsai Y C , Kendrick M I , Kass E H
Published: January 2020

It has been postulated that toxic shock syndrome may be mediated by the hydrolysis of certain tampon fibers by bacteria of the female genital tract, leading to the release of glucose that would in turn serve as a substrate for the multiplication of staphylococci producing the toxic shock marker protein (TSST-1). We sought cellulolytic organisms among microorganisms isolated from the female genital tract throughout the menstrual cycle. A total of 288 aerobic and facultative vaginal isolates from 13 healthy female volunteers, aged 18 to 25, and 57 anaerobes from the same sources were screened for cellulase activity. No evidence of production of glucose or degradation of cellulose was found; hence, none of the strains could be described as cellulolytic. A total of 44 organisms (12.7%) showed weak endoglucanase activity as evidenced by minimal changes in the viscosity of the cellulose substrate, but this activity was not reproducible in all of the strains and was inconstantly observed on repeated examination. Five strains of Staphylococcus aureus isolated from cases of toxic shock syndrome also showed no cellulase activity. Cellulase activity does not appear to be a frequent or regular feature of the microflora of the human female genital tract.
Relationship of cell surface morphology and composition of Streptococcus salivarius K+ to adherence and hydrophobicity
Authors: Weerkamp A H , van der Mei H C , Slot J W
Published: January 2020

The cell surfaces of a range of variants of Streptococcus salivarius HB, altered in cell wall antigen composition, were compared with those of the parent with respect to adherence, ability to adsorb to hexadecane, morphology, and exposure of lipoteichoic acid (LTA). Adherence to host surfaces was measured by using both saliva-coated hydroxyapatite beads and tissue-cultured HeLa cells, and interbacterial adherence was measured by using Veillonella alcalescens V1 cells. Progressive loss of the protease-sensitive fibril classes was generally associated with decreasing ability to adsorb to hexadecane. However, increased exposure of protein antigen C (AgC) increased the apparent hydrophobicity of the cell. This correlated with the finding that AgC was the most hydrophobic of the solubilized fibrillar cell wall antigens. Collectively, this demonstrates that adsorption to hydrophobic ligands is directly related to the density of the fibrillar layer on the cells and the properties and surface exposure of specific fibril classes. The involvement of hydrophobic interactions in AgC-associated attachment was suggested by its sensitivity to low levels of the hydrophobic bond-breaking agent tetramethyl urea, although the reduction was not to the level of adherence observed with strains lacking AgC. However, hydrophobicity was less essential to other adherence reactions. Circumstantial evidence, including immunoelectron microscopy, showing that LTA was virtually absent from the fibrillar layer, whole-cell enzyme-linked immunosorbent assay, suggesting that surface exposure of LTA related inversely to the density of the fibrillar layer, and agarose gel electrophoresis, showing that LTA was not specifically associated with protein fibrillar antigens, strongly suggested that LTA does not confer hydrophobic properties to these cells and is not involved in adherence reactions associated with the cell wall protein antigens.
Resistance to pesticin, storage of iron, and invasion of HeLa cells by Yersiniae
Authors: Sikkema D J , Brubaker R R
Published: January 2020

The independent abilities of Yersinia pestis to absorb exogenous pigments including hemin and Congo red (Pgm+) and to produce the bacteriocin pesticin with genetically linked invasive enzymes (Pst+) are established virulence factors of the species. Pst- Pgm+ strains of Y. pestis are sensitive to pesticin (Psts), and mutation of these isolates to pesticin resistance (Pstr) is known to result in concomitant conversion to Pgm-. Wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica are Pgm- but may be Psts; mutation of the latter to Pstr also results in avirulence. In this study, typical Pgm- mutants of Y. pestis exhibited a dramatic nutritional requirement at 37 degrees C but not 26 degrees C for iron which could be fulfilled by either Fe3+ or hemin. Iron privation of Pgm- yersiniae resulted in formation of osmotically stable spheroplasts similar to those previously observed after exposure of Psts bacteria to pesticin. At 37 degrees C, Pgm+ organisms rapidly overgrew initially predominant Pgm- populations in iron-deficient medium. However, Pgm-isolates could undergo a second mutation that permitted successful competition with Pgm+ cells in this environment. The mutation to Pstr in Y. pseudotuberculosis and Y. enterocolitica did not promote a similar requirement for iron but rather prevented these organisms from penetrating HeLa cells. The ability to invade these nonprofessional phagocytes was not shared by Pgm+ or Pgm- cells of Y. pestis.
Variable severity and Ia antigen expression in streptococcal-cell-wall-induced hepatic granulomas in rats
Authors: Allen J B , Wilder R L
Published: January 2020

We have previously reported that a single intraperitoneal injection of an aqueous suspension of group A streptococcal cell wall (SCW) fragments induces extensive hepatic granulomas in LEW/N female rats, but not in F344/N female rats. To further understand the mechanisms underlying these differences, we compared granuloma development and class II major histocompatibility complex antigen (Ia) expression in histocompatible LEW/N, F344/N, and CAR/N female rats in response to SCW fragments of four different average molecular sizes. In LEW/N female rats, the smallest fragments (less than 5 megadaltons) induced the most severe hepatic inflammatory disease, with development of widespread granulomas composed of macrophages, lymphocytes, and a peripheral rim of fibroblasts. The largest fragments (greater than 500 megadaltons) induced equivocal disease. Fragments of intermediate size induced granulomas of intermediate severity. The extent of granuloma development, the intensity of Ia antigen expression, and the amount of SCW antigen deposited in the liver qualitatively paralleled each other. In contrast, injection of the most granulomagenic SCW fragments into F344/N and CAR/N rats did not induce granulomas. Although these rat strains are histocompatible with the LEW/N (i.e., RTL.1) strain, hepatic Ia antigen expression in these strains was not increased significantly above basal levels. The amount of SCW antigen in the livers of the resistant rat strains appeared similar to the amount in the susceptible LEW/N strain. These data indicate that granuloma development is dependent on the size of the SCW fragment and host genetic background and that Ia expression directly parallels the severity of the hepatic disease. In addition, the data suggest that non-major histocompatibility complex genetic loci play a major role in regulating the development of the hepatic disease.
Metabolism of glycosylsucrose by oral microorganisms and its hydrolysis by Streptococcus salivarius fructosyltransferase
Authors: Hojo S , Mitsutomi M , Yamada T
Published: January 2020

Resting-cell suspensions of oral microorganisms grown in sucrose were studied for the production of acid from glucosylsucrose and maltosylsucrose. Most oral microorganisms fermented these sugars to only a limited extent. Streptococcus salivarius, however, metabolized glucosylsucrose as well as sucrose. We therefore looked for a specific enzyme in S. salivarius which was capable of hydrolyzing glucosylsucrose. Fructosyltransferase and invertase were purified from S. salivarius 13419, and the substrate specificities and hydrolytic activities of these enzymes were determined. Purified fructosyltransferase catalyzed fructan synthesis from glucosylsucrose or maltosylsucrose, whereas purified invertase barely hydrolyzed these sugars. These results suggest that the high fermentative efficiency of glycosylsucrose by S. salivarius is due to the hydrolysis of these sugars by fructosyltransferase, but not by invertase. The partially purified fructosyltransferases of Actinomyces viscosus NY1 and Streptococcus mutans NCIB 11723 catalyzed fructan synthesis from glucosylsucrose or maltosylsucrose. The fructosyltransferases of these oral microorganisms are also responsible for the hydrolysis of glycosylsucrose.
Serum-mediated suppression of lymphocyte transformation responses in coccidioidomycosis
Authors: Cox R A , Pope R M
Published: January 2020

Lymphocyte transformation (LT) responses to coccidioidin (CDN) and spherulin were suppressed in 11 (73%) of 15 patients with active coccidioidomycosis when their mononuclear cells were assayed in autologous serum as compared to serum from healthy, CDN skin test-positive subjects. Suppressed LT responses were specific for Coccidioides immitis antigens in 7 (64%) of the 11 patients. Immunoaffinity chromatography of patient sera with Staphylococcus protein A adsorbed the suppressor component(s) and thereby established that suppression was attributed to immunoglobulin G, either alone or complexed with antigen. The possibility that suppression was mediated by immune complexes was examined by adding complexes formed in vivo or in vitro to mononuclear cell cultures of healthy CDN-reactive persons before LT assays. Although complexes prepared in this manner were reactive in an enzyme-linked immunosorbent assay designed to detect Coccidioides antigen-specific immune complexes, no suppression of LT responses was observed. We conclude that serum-mediated suppression of LT responses in coccidioidomycosis is attributed to monomeric and not immune-complexed immunoglobulin G antibody.
Investigation of synthetic Escherichia coli heat-stable enterotoxin as an immunogen for swine and cattle
Authors: Frantz J C , Bhatnagar P K , Brown A L , Garrett L K , Hughes J L
Published: January 2020

In its native form Escherichia coli heat-stable enterotoxin (STa) is nonantigenic; however, neutralizing antibodies are elicited in animals vaccinated with toxin-carrier conjugates. To study the immunogenicity of STa, peptides STa1-18 and STa5-18 were synthesized, characterized, and conjugated to carrier proteins. Pregnant gilts and heifers were hyperimmunized with the respective conjugates. Following parturition neonates were challenged with virulent E. coli (K99+ STa+). Peptides coupled to ovalbumin and emulsified with Freund adjuvant elicited antibodies that neutralized toxin-induced fluid accumulation in suckling mice. Peptides coupled to particulate carriers, with or without muramyl dipeptide adjuvant, failed to induce a measurable response. Peak antibody levels in sera were observed following three doses of conjugate and persisted for several weeks. The serological response in cattle was superior to that observed in swine; however, antibody levels in porcine colostrum were higher than those observed in cattle. Clinical observations of neonates from vaccinated dams indicated that passively obtained antibody provided partial protection from disease, but not as complete as that demonstrable with whole cell bacterins that induce antibody to pili. However, the data suggest the potential for utility of synthetically prepared antigens.
Plasmid-mediated resistance to phagocytosis in Yersinia enterocolitica
Authors: Lian C J , Hwang W S , Pai C H
Published: January 2020

Results of our previous studies have shown that the chemiluminescence response of human neutrophils (polymorphonuclear leukocytes [PMNs]) is inhibited by plasmid-mediated cell surface components from Yersinia enterocolitica. In this study we examined the susceptibility to phagocytosis of Y. enterocolitica cells with or without plasmid-mediated surface structure and the effect of isolated outer membrane fragments on phagocytosis of Escherichia coli by PMNs in vitro. Y. enterocolitica cells with expressed plasmid-mediated surface structure were much less sensitive to ingestion by PMNs than those without it, and the resistance to phagocytosis was readily eliminated in a dose-dependent fashion by pronase treatment of whole cells, which was shown to remove plasmid-encoded outer membrane proteins. Ingestion and intracellular killing of E. coli were inhibited significantly in the presence of isolated outer membrane fragments derived from plasmid-bearing Y. enterocolitica cells. To assess the interaction of Y. enterocolitica with phagocytic cells in vivo, two isogenic strains of Y. enterocolitica, differing only in the presence or absence of the virulence plasmid, were inoculated intradermally into the backs of rabbits; and tissue sections obtained at 12 h postinoculation were examined by light and electron microscopy. The plasmidless strain was found almost entirely in PMNs or mononuclear cells. In contrast, the plasmid-bearing strain was found to be surrounded by, or interspersed with, PMNs and mononuclear cells; but most bacteria were extracellular, with little evidence of phagocytosis. These results suggest that plasmid-mediated cell surface components of Y. enterocolitica act as antiphagocytic factors, thus facilitating the survival and proliferation of the organism in the host tissue.
Cloning and expression of the gene for an immunoglobulin G Fc receptor protein from a group A streptococcus
Authors: Heath D G , Cleary P P
Published: January 2020

DNA containing the 5' end of the M-12 structural gene was used as a probe in colony hybridizations in an attempt to clone the M-76 gene from an M-type 76 group A streptococcal strain. A single positive colony was detected, and Southern hybridization analysis of plasmid DNA isolated from this colony indicated that the insert DNA had homology to the 5' end of the M-12 structural gene. Subclones were constructed to define the limits of the M-76 gene, and sonicates of these subclones were reacted with M-76-specific antiserum in immunodiffusion. A sonicate of one subclone, JM83(pDH56), reacted strongly with the M-76-specific antiserum but also reacted with preimmune rabbit serum. Protein expressed from this subclone bound immunoglobulin from horse and pig, as well as human myeloma immunoglobulin G (IgG) representing all four subclasses and purified human IgG Fc fragments. This indicated that JM83(pDH56) expressed a protein with characteristics previously attributed to the IgG Fc receptor protein from group A streptococci. Western blot analysis indicated that the cloned IgG Fc receptor protein had a molecular weight of approximately 29,000. Binding studies showed that the Fc receptor gene is expressed by the M-type 76 strain from which it was cloned and by an M- variant.
Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae
Authors: Clark V L , Campbell L A , Palermo D A , Evans T M , Klimpel K W
Published: January 2020

Neisseria gonorrhoeae is generally considered to be an obligate aerobe; it can, however, grow in the absence of oxygen by anaerobic respiration by using nitrite as a terminal electron acceptor. The outer membrane protein compositions of aerobically and anaerobically grown N. gonorrhoeae strains were compared by one- and two-dimensional polyacrylamide gel electrophoresis. Anaerobically grown strains expressed at least three proteins (Pan 1 to Pan 3) at much higher levels than did aerobically grown cells. Conversely, at least five other proteins (Pox 1 to Pox 5) were found to be expressed at significantly higher levels in aerobically grown cells. None of the Pan or Pox proteins were heat modifiable, and none of the heat-modifiable protein IIs or other major outer membrane proteins (protein I, protein III, pilin, or H-8 protein) were significantly altered in expression by anaerobic growth. There were also no apparent differences in lipopolysaccharide composition in aerobically and anaerobically grown gonococci. The regulation of protein expression by oxygen availability suggests that anaerobic growth is a physiologically significant state for this organism.
Characterization of a third-order suppressor T cell (Ts3) induced by cryptococcal antigen(s)
Authors: Khakpour F R , Murphy J W
Published: January 2020

Previous studies from our laboratory have shown that a high dose of cryptococcal culture filtrate antigen (CneF) administered intravenously induces a complex suppressor cell cascade which down-regulates the cell-mediated immune response to Cryptococcus neoformans antigens. The primary objective of this investigation was to determine whether a suppressor cell induced by immunization is required for efferent suppression of the cryptococcal delayed-type hypersensitivity (DTH) response. Our approach to this problem was to immunize CBA/J mice with CneF emulsified in complete Freund adjuvant and then 6 days later to collect spleen cells from the immunized mice and adoptively transfer these cells along with C. neoformans-specific second-order suppressor T cells (Ts2) to naive syngeneic recipients at the time of footpad challenge of the recipients with CneF. To establish which populations of cells in the spleens of immunized mice play a suppressive role, mass cytolysis with specific antibodies and complement was performed before the spleen cells were transferred to naive animals. Since the phenotype of the cells responsible for the transfer of the cryptococcal DTH response had not been completely determined, we first demonstrated that the cells responsible for DTH were L3T4+ Lyt-2- cells. Subsequently, we established that a Thy-1+ L3T4- Lyt-2+ I-J+ cell population induced by immunization was required along with C. neoformans-specific Ts2 cells for efferent suppression of the cryptococcal DTH response. In addition, we demonstrated that the suppressor cells in the immune cell population were derived from cyclophosphamide-sensitive precursors. These data indicate that a third suppressor cell population is required for efferent suppression of the cryptococcal DTH response. As in the azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl hapten suppressor models, the Ts2 cells in the circuit mediate their effects through this third suppressor component. Since the mode of induction and the phenotype of the third C. neoformans-specific suppressor cells are similar to those reported for Ts3 cells in other antigen-specific suppression models, we referred to this third suppressor cell in the C. neoformans-specific suppressor cell cascade as a Ts3 cell.
Biological and biochemical characterization of tunicamycin-resistant Leishmania mexicana: mechanism of drug resistance and virulence
Authors: Kink J A , Chang K P
Published: January 2020

A parasitic protozoan, Leishmania mexicana amazonensis, was previously made resistant to tunicamycin (J.A. Kink and K.-P. Chang, Proc. Natl. Acad. Sci. USA 84:1253-1257, 1987). In the present study, six different tunicamycin-resistant variants were biologically and biochemically compared with their parental wild type to further delineate the mechanism of tunicamycin resistance and that of their virulence observed. In contrast to their parental wild type, all tunicamycin-resistant variants were found to grow and differentiate in tunicamycin-containing medium. The 50% lethal doses of tunicamycin for variants resistant to 10 or 80 micrograms of tunicamycin per ml were 20- and 100-fold higher, respectively, than that of the wild type. Specific activity of the microsomal N-acetylglucosamine-1-phosphate transferase was 4- to 12-fold higher in the tunicamycin-resistant cells than in their parental wild type and tunicamycin-sensitive revertants. The level of the enzyme activity is proportional to the degree of drug resistance. Inhibition kinetics studies showed that the enzyme from all groups was equally sensitive to the drug, with a 50% effective concentration of 1 to 1.3 micrograms of tunicamycin per ml. Thus, tunicamycin resistance of the variants is caused primarily by an increased level of their enzyme without alteration of its structure. Protein glycosylation determined by the incorporation of 2-D-[3H]mannose was about twofold higher in the tunicamycin-resistant variants than in their parental wild type. The increased glycosyltransferase activity in the latter apparently renders their protein glycosylation insensitive to the inhibition by tunicamycin. A major membrane glycoprotein of 63 kilodaltons (gp63) on the leishmania surface was found to be about threefold higher in the tunicamycin-resistant variants than in the wild type, as determined by immunoprecipitation with a monoclonal antibody specific for this antigen. Tunicamycin treatment of the wild type and tunicamycin-resistant variants caused changes in the electrophoretic mobility of this molecule, indicating a higher degree of its glycosylation in the latter cells. The tunicamycin-resistant variants parasitized macrophages in vitro more effectively than did the wild type, accounting for their virulence seen in mice. Thus, a high level of the glycosyltransferase enables the tunicamycin-resistant cells not only to overcome the inhibitory effect of tunicamycin on protein glycosylation but also to express their virulence, possibly by regulating N glycosylation of leishmanial proteins critical for leishmanias to establish intracellular parasitism.
Activation and binding of C3 by Candida albicans
Authors: Kozel T R , Brown R R , Pfrommer G S
Published: January 2020

Interaction with components of the complement system is an important aspect of the pathogenesis of infection by Candida albicans. The key role of C3 as an opsonic ligand and as an element in amplification of complement activation led us to examine several factors that influence the activation and binding of C3 cleavage fragments to the yeast. Activation and binding of C3 were determined by use of normal human serum containing 125I-labeled C3. Incubation of yeast-phase cells in 20% serum led to deposition of 2.5 X 10(5) to 3.0 X 10(5) molecules of C3 per yeast cell. Binding of C3 was absent in serum that was heat inactivated for 30 min at 37 degrees C or in serum that was chelated with EDTA. Chelation of serum with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] reduced binding of C3 fragments by 31%. These results suggest that the alternative complement pathway is the primary mechanism for activation and binding of C3 fragments to C. albicans. Bound C3 could be partially removed (50%) by treatment with 1.0 M hydroxylamine. In contrast, 1.0 M hydroxylamine removed 98% of the C3 fragments bound to encapsulated Cryptococcus neoformans. These results suggest that ester bonds are the primary mechanism for binding C3 to C. neoformans, whereas binding of C3 to C. albicans involves both ester and amide bonds. Monoclonal antibodies specific for C3c and an iC3b neoantigen were used to identify the fragment of C3 that was bound to C. albicans. The results showed that the primary fragment bound to the yeast was C3b. An examination of the kinetics of activation and binding of C3 fragments showed that activation and binding were very rapid. Near-maximal binding occurred after a 2.5- to 5-min incubation period. In contrast, activation and binding of C3 fragments to C. neoformans proceeded at a slower rate, with maximal binding requiring 10 to 20 min. These results indicate that activation and binding of C3 fragments by the yeasts C. albicans and C. neoformans differ in several important characteristics.
Monoclonal antibodies to the extracellular glucosyltransferases from Streptococcus sobrinus 6715
Authors: McCabe M M , Alberts M , Stein J
Published: January 2020

Murine monoclonal antibodies (MAbs) were raised against the glucosyltransferases (GTFs) of Streptococcus sobrinus 6715. The antibody panels included MAbs raised against the primer-independent, soluble product enzyme (GTF-Si) which did not cross-react with other GTFs, as well as MAbs raised against the primer-dependent, soluble product enzyme (GTF-Sd) which recognized both GTF-Si and GTF-Sd, thus indicating that these catalytically distinct enzymes share epitopes. MAbs raised against GTF-I recognized several forms of GTF-I and did not cross-react with the GTF-S enzymes. None of the MAbs recognized the major glucan-binding protein of S. sobrinus. Two MAbs inhibited glucan synthesis, one blocking primer synthesis by GTF-Si by 89% and the second inhibiting that by GTF-I by 92%.
Distribution of 3H-labeled staphylococcal alpha-toxin and a toxin fragment in mice
Authors: Blomqvist L , Appelgren L E , Thelestam M
Published: January 2020

Staphylococcal alpha-toxin and a toxin fragment were labeled with N-succinimidyl[2,3-3H]propionate. The labeled compounds retained greater than 95% biological activity. The distribution of labeled staphylococcal alpha-toxin and alpha-toxin fragment after intravenous administration to BALB/c mice was studied with whole-body and microautoradiography. The animals were divided into three groups that received (i) labeled alpha-toxin only, labeled alpha-toxin after prior injection of unlabeled fragment, or labeled fragment only. After 5 min, the distribution patterns were similar in groups 1 and 2, with the highest amounts of radioactivity found in the blood vessels, liver, spleen, lungs, and kidneys, whereas the labeled fragment alone showed no initial accumulation in the lungs. The kidneys continued to show a high concentration of radioactivity, whereas the levels at 60 min had decreased in the other organs. The toxin showed continued stable binding to the proximal tubuli, whereas the toxin fragment seemed to dissociate and was found only in small amounts in the glomeruli. No radioactivity was found in the central nervous system.
Role of Peyer's patch in the intestinal immune response to cholera toxin in enterically immunized rats
Authors: Enders G A , Delius M , Ballhaus S , Brendel W
Published: January 2020

Cholera toxin has been widely used to obtain insight into the cellular dynamics of the antigen-specific mucosal immune response. The present study was undertaken to clarify the influence of the organized intestinal lymphoid tissue (Peyer's patches [PP]) on the distribution of anti-cholera-toxin-containing cells (ACC) after intraperitoneal immunization and intraduodenal challenge with purified cholera toxin. This was done in rats which were surgically deprived of all visible PP. In comparison with sham-operated animals, each PP-deprived rat had nearly the same amount of ACC in the spleen, the mesenteric lymph nodes, and, surprisingly, the thoracic duct lymph. In contrast, the ACC in the duodenum, the jejunum, and the ileum of each PP-deprived animal were drastically reduced. Therefore the PP are suggested as an important organizing structure for the buildup of a local antigen-specific immune response.
Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells
Authors: Müller I , Cobbold S P , Waldmann H , Kaufmann S H
Published: January 2020

The resistance of mice against Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells was studied. Thymectomized mice were treated with rat monoclonal antibodies against the L3T4 or Lyt-2 molecule to selectively eliminate the respective T-cell subset. In both L3T4+ and Lyt-2+ T-cell-depleted mice, resistance against subsequent infection with M. tuberculosis was markedly impaired compared with that in untreated controls, with L3T4+ T-cell-depleted mice showing more pronounced effects. Simultaneous depletion of L3T4+ and Lyt-2+ T cells did not further exacerbate infection. These findings suggest that both L3T4+ and Lyt-2+ T cells are involved in the acquisition of resistance against tuberculosis.
Delta-like toxin produced by coagulase-negative staphylococci is associated with neonatal necrotizing enterocolitis
Authors: Scheifele D W , Bjornson G L , Dyer R A , Dimmick J E
Published: January 2020

Necrotizing enterocolitis (NEC) is a serious neonatal disorder of uncertain cause, although fecal bacteria have been implicated in some outbreaks. We examined coagulase-negative staphylococci (CONS) as possible etiologic agents. In our unit, CONS colonized the bowels of most infants studied, including 46% of 70 NEC cases (mean concentration, 10(9.1) CFU/g of stool). Over 90% of tested isolates produced a hemolysin resembling delta toxin of Staphylococcus aureus. Toxin purified from a NEC-associated isolate of Staphylococcus epidermidis resembled reference delta toxin from S. aureus in size, biologic properties, and antigenicity. This delta-like toxin was enteropathic, causing mucosal necrosis and hemorrhage in injected loops of the bowels of infant rats. Adjacent, nonexposed bowel remained normal, as did loops injected with lecithin-neutralized toxin. Using a new enzyme-linked immunosorbent assay (ELISA), we detected delta-like toxin in the stools of 11 of 35 infants colonized with CONS positive for delta-like toxin (Tox+). Positive tests were strongly associated with NEC. Of 18 cases with Tox+ CONS, 10 were positive (56%), whereas only 1 of 17 control infants so colonized was positive (6%, P = 0.002). In NEC patients, the mean fecal toxin concentration was 1,012 ng/g. Toxicity to fibroblasts was demonstrable in filtrates of each of six ELISA-positive samples tested but was absent in all five ELISA-negative samples tested. We conclude that delta-like toxin is elaborated in the bowels of some infants with Tox+ CONS, and its association with NEC suggests that such CONS are enteropathic. In our unit, this mechanism was apparent in 23% of 44 recent cases of endemic NEC.
Purification of actin from Candida albicans and comparison with the Candida 48,000-Mr protein
Authors: Fiss E , Buckley H R
Published: January 2020

Actin was purified from Candida albicans cells by affinity chromatography by DNase-Sepharose and was recognized by immunoblotting with monoclonal antibody directed against chick muscle actin. The C. albicans 48-kilodalton protein recognized by sera from patients with invasive candidiasis was shown by DEAE chromatography and immunoblotting not to be identical with the purified C. albicans actin.
Mapping of the T-cell recognition sites of Pseudomonas aeruginosa PAK polar pili
Authors: Smart W , Sastry P A , Paranchych W , Singh B
Published: January 2020

The polar pili of Pseudomonas aeruginosa consist of a subunit protein, pilin, which is a 144-residue polypeptide that contains a hydrophobic N-terminal region and eight hydrophilic regions distributed throughout the remainder of the molecule. T cells from mice immunized with pili or whole bacteria gave good pilus-specific T-cell proliferation responses. To delineate the T-cell antigenic regions of the pilin, T-cell blasts were generated from lymph nodes of pilus-primed BALB/c mice. These blasts were tested in vitro in T-cell proliferation assays for reactivity against the fragments of the pilin subunit prepared by enzymatic digestion. Citraconylation followed by trypsin digestion (cT) of the pilin subunit cleaved the protein into four fragments, cTI (residues 1 to 30), cTII (residues 31 to 53), cTIII (residues 54 to 120), and cTIV (residues 121 to 144). The ability to stimulate the T cells was found to reside in the cTI and cTIII regions, but not in the cTII or cTIV regions. A subfragment of cTIII, containing residues 82 to 104, was identified as the major T-cell recognition site within the cTIII region of the pilin molecule. A cross-reactivity was observed between pili from two strains of P. aeruginosa, namely, PAK and PAO, at the T-cell level. This cross-reactivity probably resulted from the sequence homology in the hydrophobic N-terminal region of these two molecules.
Partial purification of a bacterial lectinlike substance from Eikenella corrodens
Authors: Yamazaki Y , Ebisu S , Okada H
Published: January 2020

A bacterial lectinlike substance, which is considered to participate in the adherence of Eikenella corrodens to various host cells, was purified from E. corrodens cells. The substance was extracted in 1% Triton X-100 with sonication from the cell envelope of E. corrodens 1073 and partially purified by galactosamine affinity chromatography and gel filtration chromatography based on its hemagglutination (HA) activity. The lectinlike substance was purified about 256-fold as evaluated by its specific HA activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified lectinlike substance (PPL) produced a single protein band of large molecular weight when it was applied to the gel without the addition of beta-mercaptoethanol and heating. Chemical analysis showed that PPL contained 14.4 micrograms of hexose per 100 micrograms of protein and that it did not contain muramic acid, glucosamine, or 2,6-diaminopimelic acid, which are characteristic of peptidoglycans. The HA activity of PPL was inhibited by EDTA but restored by adding Ca2+. The HA activity was remarkably inhibited by sugars containing N-acetyl-D-galactosamine and D-galactose. These results indicate that the lectinlike substance on the E. corrodens cells is an essential factor for the adherence to host cells.
Enterotoxigenic Escherichia coli diarrhea in an endemic area prepares the intestine for an anamnestic immunoglobulin A antitoxin response to oral cholera B subunit vaccination
Authors: Holmgren J , Svennerholm A M , Gothefors L , Jertborn M , Stoll B
Published: January 2020

We examined whether infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-labile enterotoxin (LT) can prime the gut immune system to respond more efficiently to the immunologically related cholera B subunit component of a recently developed oral B subunit-whole-cell cholera vaccine (B-WCV). Nine Bangladeshi adults who had been hospitalized for watery diarrhea caused by LT-producing ETEC were given a single oral immunization with B-WCV on day 28 after hospital admission. The vaccine preparation used was adjusted to contain a lower-than-usual dose of B subunit, which had been found in previous studies to elicit a significant gut mucosal immunoglobulin A antitoxin response mainly in individuals with previous toxin-specific priming of their gut immune system. For comparison, nine patients convalescing from severe cholera disease and eight healthy subjects with no recent history of either cholera or ETEC infection were given the same oral vaccination with B-WCV. Vaccination in the ETEC convalescents induced an immunoglobulin A antitoxin response in intestinal lavage fluid which was comparable with that in the vaccinated cholera convalescents and superior to that in the vaccinated, previously uninfected controls. By contrast, only the cholera patients responded with anamnestic-type anti-cholera lipopolysaccharide antibody titer rises in the intestine after vaccination. These data support the specificity of the anamnestic anti-cholera toxin response in the ETEC patients after vaccination with cholera B-WCV.
Prevention of peroral and congenital acquisition of Toxoplasma gondii by antibody and activated macrophages
Authors: Eisenhauer P , Mack D G , McLeod R
Published: January 2020

Intramuscular administration of Toxoplasma gondii lysate antigens to mice produced titers of T. gondii-specific antibody (measured by Sabin-Feldman dye test) greater than or equal to 1:1,024 in their sera. Intravenous administration of heat-killed Propionibacterium acnes to mice produced peritoneal macrophages with enhanced microbicidal capacity against T. gondii. Mice with high antibody titers or activated peritoneal macrophages or both had reduced numbers of Toxoplasma cysts in their brains 30 days after peroral challenge. Specific antibody and activated macrophages appeared to act together to significantly (P = 0.01) reduce the numbers of Toxoplasma cysts. A reduction in tissue infection as a result of these treatments was also demonstrated by subinoculation of brain tissue. A high antibody titer alone did not protect against congenital infection. Mice treated with P. acnes delivered reduced numbers of T. gondii-infected pups (P greater than 0.05). Treatment that produced high titers of Toxoplasma antibody and activated macrophages provided significant protection against congenital infection (P less than 0.05).
Isolation of a chymotrypsinlike enzyme from Treponema denticola
Authors: Uitto V J , Grenier D , Chan E C , McBride B C
Published: January 2020

A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, alpha 1-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50 degrees C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.
Chronic giardiasis in B-cell-deficient mice expressing the xid gene
Authors: Snider D P , Skea D , Underdown B J
Published: January 2020

The role of antibody in immunity to Giardia muris infection was investigated by studying B-cell-deficient CBA/N mice expressing the xid gene. After gastric administration of infective G. muris cysts, CBA/N male and female mice developed prolonged G. muris infection, whereas BALB/c mice eliminated their infection in 6 to 8 weeks. Male F1 progeny obtained from matings between female CBA/N mice and male BALB/c mice expressed the xid gene and developed prolonged infections. In contrast, all other F1 progeny of CBA/N and BALB/c matings, which did express the xid gene, eliminated G. muris. The link between the xid gene and prolonged infection was confirmed by studies of C57BL/6 mice congenic for the xid gene. When compared with BALB/c or F1 mice, CBA/N mice produced large quantities of immunoglobulin A (IgA) anti-G. muris antibody in serum and gut secretions during prolonged infection. Serum IgG anti-G. muris antibody levels were reduced in CBA/N and F1 male mice that expressed the xid gene. The inability of xid mice to eliminate G. muris is consistent with the importance of antibody in the development of immunity to G. muris. We hypothesize that mice bearing the xid gene fail to produce IgA antibody of appropriate specificity to an antigen or antigens whose recognition by antibody is critical for successful elimination of the parasite.