bluuebunny/crossref_metadata_2025 huggingface dataset:
Salmonella choleraesuis strains deficient in O antigen remain fully virulent for mice by parenteral inoculation but are avirulent by oral administration
Authors: Nnalue N A , Lindberg A A
Published: January 2020

O-antigen-deficient derivatives of two mouse-virulent strains of Salmonella choleraesuis (serogroup C1; O-6,7) were constructed by transduction of a long deletion of the rfb operon. Strains SN36 and SN57 were derived from the smooth ancestor SL2824, while SN37 was derived from the smooth ancestor SL2840. These rfb deletion derivatives (rfb strains) had typical bacteriophage sensitivity patterns of "rough" Salmonella strains and were at least 200,000 times more sensitive to serum than their smooth ancestors. Lipopolysaccharides (LPS) extracted from them consisted only of two low-molecular-weight bands and lacked the ladderlike pattern of bands seen in the LPS of their smooth ancestors. The LPS from the rfb strains did not react in an enzyme immunoassay with any of three monoclonal antibodies directed against different epitopes of the O-6,7 antigen but reacted well with at least one of three monoclonal antibodies specific for core epitopes. The data were consistent with inability of these strains to synthesize O-specific chains and showed that the LPS extracted from SN57 was of chemotype Ra and that from SN36 was of chemotype Rb1, while that of SN37 consisted of a mixture of the two chemotypes. The virulence of these strains was tested by various routes in BALB/c mice. All three O-antigen-deficient derivatives were about as virulent as their "smooth" ancestors by the intraperitoneal and intravenous routes (50% lethal dose, 20 to 700 bacteria) but, unlike their ancestors, were avirulent by the oral route (50% lethal dose, greater than or equal to 5 x 10(9) bacteria). This suggests that the major role of smooth LPS in the mouse virulence of S. choleraesuis is to facilitate survival in the gastrointestinal tract and eventual entry into deeper tissues.
Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis
Authors: Molina N C , Parker C D
Published: January 2020

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.
Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa
Authors: Saiman L , Cacalano G , Prince A
Published: January 2020

Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.
Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells
Authors: Suhrbier A , Winger L A , Castellano E , Sinden R E
Published: January 2020

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.
Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae
Authors: Galán J E , Timoney J F
Published: January 2020

Erysipelothrix rhusiopathiae is a primary pathogen of swine and turkeys and sporadic cause of disease in a variety of other hosts, including humans. A genomic library of the highly virulent strain of E. rhusiopathiae E1-6P was constructed in the expression-cloning vector lambda gt11 and screened with serum from a pig convalescent from an E. rhusiopathiae experimental infection. Immunoreactive clones were screened for their ability to protectively immunized mice. Two clones, lambda gt11/ersA and lambda gt11/ersB, were obtained that protected mice against challenge with E. rhusiopathiae E1-6P. Antisera against the recombinant clones reacted with polypeptides of molecular weights 66,000, 64,000, and 43,000 in detergent-solubilized surface antigen preparations and whole-cell lysates of E. rhusiopathiae. These polypeptides were also the major antigens recognized by convalescent pig serum when reacted with the same preparations. Western immunoblot and Southern blot analysis revealed that the cloned genes and gene products were present in all of the E. rhusiopathiae strains tested.
Cyclosporin A treatment converts Leishmania donovani-infected C57BL/10 (curing) mice to a noncuring phenotype
Authors: Adinolfi L E , Bonventre P F
Published: January 2020

Cyclosporin A prevents visceralization of Leishmania major infection of BALB/c mice (N. C. Behforouz, C. D. Wenger, and B. A. Mathison, J. Immunol. 136:3067-3075, 1986; W. Solbach, K. Forberg, E. Kammerer, C. Bogdan, and M. Rollinghoff, J. Immunol. 134:702-707, 1986). We report that cyclosporin A exacerbates disseminated leishmaniasis caused by L. donovani in C57BL/10 mice. Normal mice challenged with 5 x 10(6) amastigotes intravenously cleared the infection within several months by spontaneous acquisition of cell-mediated immunity. In contrast, cyclosporin A administered daily intraperitoneally at a dose of 1.25 mg per mouse prevented development of curative immunity and converted C57BL/10 (curing) mice to a noncuring phenotype. A rationale for the contrasting effects of cyclosporin A in the two murine models of leishmaniasis is provided.
Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli
Authors: Phelps C J , Lyerly D L , Johnson J L , Wilkins T D
Published: January 2020

Cloned fragments constituting the 8.1-kb toxin A gene of Clostridium difficile were used to reconstruct the intact gene. The recombinant toxin expressed in Escherichia coli was cytotoxic, enterotoxic, and lethal. In addition, toxic lysate caused hemagglutination of rabbit erythrocytes. The toxic activities were inhibited by antibody specific for toxin A. Our findings demonstrate that the biological activities exhibited by native toxin A are functions of a single protein encoded by the 8.1-kb toxin A gene, independent of any other C. difficile gene products.
Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes
Authors: Ogata M , Pastan I , FitzGerald D
Published: January 2020

Pseudomonas exotoxin (PE) is a protein toxin composed of three structural domains. In its native form, the toxin is a 66,000-Mr proenzyme that must be activated to express full ADP-ribosylating activity. To study the process of activation and accompanying conformational changes, we have isolated 10 monoclonal antibodies to a 40,000-Mr fragment of the toxin (PE40) that exhibits full enzyme activity but lacks the toxin's cell-binding domain and contains amino acids 253 to 613 (comprising domains II, Ib, and III). By using mutant PE molecules in which all of domain I and portions of domains II, Ib, and III were deleted, the locations of the epitopes for each of the antibodies were determined. Eight of these monoclonal antibodies were further characterized. Of these eight, all reacted with soluble PE40 and an interleukin-2-PE40 conjugate, but only two reacted strongly with native soluble PE. However, all eight reacted with PE after it had been immobilized on nitrocellulose or after it had been activated to express full ADP-ribosylating activity. Antibodies were also assessed for their ability to neutralize the cytotoxic activity of either PE or interleukin-2-PE40. These antibodies should be useful as probes for monitoring the activation and processing of PE that occur during endocytosis and in determining the location of epitopes that are important for toxin activity.
Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene: identification of a species-specific sequence
Authors: Parra C A , Londoño L P , Del Portillo P , Patarroyo M E
Published: January 2020

A rabbit polyclonal antiserum exhibiting a specific recognition pattern for Mycobacterium tuberculosis proteins was used to screen an M. tuberculosis genomic library constructed in the expression vector lambda gt11. One clone, denominated C1:10, expressed M. tuberculosis-specific determinants as part of a large fusion protein with beta-galactosidase. The gene for this protein has been sequenced, and it encodes a protein of 134 amino acids (13.8 kDa) which did not display significant homology with any of the previously reported proteins in the data bases. Hybridization studies with restriction fragments of the cloned sequence revealed that it was not present in the genomes of related mycobacteria, namely, M. bovis, M. bovis BCG, M. flavescens, M. fortuitum, M. phlei, and M. vaccae. These findings suggest that we have detected a gene, or a fragment therefrom, unique for M. tuberculosis whose nucleotide and amino acid sequences could be useful tools in the design of an improved vaccine or a diagnostic method of greater accuracy for tuberculosis.
Characterization of three proteins expressed from the virulence region of plasmid pSDL2 in Salmonella dublin
Authors: Valone S E , Chikami G K
Published: January 2020

Infection of both cattle and humans with Salmonella dublin can result in septicemia and death. Like many nontyphoid Salmonella species that cause disease, S. dublin contains a cryptic plasmid (pSDL2) that is required for the full expression of virulence. Transposon mutagenesis of pSDL2 defined a 4.1-kb EcoRI region that is necessary for the development of a systemic infection in BALB/c mice. This EcoRI fragment was cloned into an expression vector (pEL11), and three proteins produced from this region with apparent molecular weights of 30,500, 76,000, and 27,000 were identified. Because bacterial proteins that play a role in virulence are often associated with the outer membrane, we were interested in establishing whether the proteins expressed from the EcoRI fragment are located in the membrane. Transposon mutagenesis of pEL11 with TnphoA defined the order of the genes along the fragment and suggested that the proteins may be exported out of the cytoplasm. Sucrose gradient cell fractionation was done to identify the cellular location of each of the three proteins. The 30-kDa protein was identified in the outer membrane fraction, and the 76-kDa protein was located in the cytosolic fraction. The 27-kDa protein was identified in both the cytosolic and the outer membrane fractions. The outer membrane contained less than 10% of the activity of enzymes known to be located in the cytoplasm, periplasm, and inner membrane. Sequence data of the 4.1-kb EcoRI region revealed that both the 30- and the 27-kDa proteins lack a typical signal sequence for export out of the cytoplasm (M. Krause, C. Roudier, J. Fierer, J. Harwood, and D. G. Guiney, Mol. Microbiol. 5:307, 1991). The outer membrane location of these proteins suggests that they may be exported out of the cytoplasm by an unusual mechanism.
Preservation of monocyte effector functions against Mycobacterium avium-M. intracellulare in patients with AIDS
Authors: Johnson J L , Shiratsuchi H , Toba H , Ellner J J
Published: January 2020

Mycobacterium avium-M. intracellulare is a frequent cause of late disseminated infection in patients with AIDS. The ability of human peripheral blood monocytes to phagocytose and kill M. avium was examined in an in vitro model. Monocytes were obtained from 13 healthy volunteers and 11 patients with AIDS, three of whom had documented disseminated M. avium infection. Monocytes were precultured for 2 days before infection with two AIDS-associated and two non-AIDS-associated strains of M. avium. Uptake of M. avium as measured by counting intracellular acid-fast bacilli did not differ among healthy subjects, patients with AIDS, or patients with AIDS and previously documented disseminated M. avium infection. Intracellular growth of M. avium was examined by a CFU assay of cell lysates from M. avium-infected monocytes after 0, 4, and 7 days of culture. Intracellular growth inhibition of M. avium at 7 days after infection was comparable between patients with AIDS and healthy donors for all M. avium strains tested. The effects of the addition of recombinant gamma interferon on M. avium uptake and intracellular growth in monocytes also were studied. Pretreatment of monocytes with gamma interferon prior to infection suppressed monocyte phagocytosis of M. avium. Continuously coculturing of monocytes with gamma interferon after infection augmented killing of M. avium among both patients with AIDS and healthy controls for three of the four strains of M. avium tested. The magnitude of this effect, however, was variable from donor to donor and strain to strain. No significant differences were noted between the growth-inhibiting abilities of gamma-interferon-treated monocytes obtained from healthy volunteers and those obtained from patients with AIDS.
Expression of Fc gamma RIII on HeLa 229 cells: possible effect on in vitro neutralization of Chlamydia trachomatis
Authors: Su H , Spangrude G J , Caldwell H D
Published: January 2020

The neutralizing activities of a murine immunoglobulin G3 (IgG3) monoclonal antibody specific for the major outer membrane protein of Chlamydia trachomatis and its monovalent Fab fragments were studied by using Syrian hamster kidney (HaK) cells and human epithelial (HeLa 229) cells. The intact IgG3 antibody was neutralizing for HaK cells but was nonneutralizing for HeLa cells. In contrast, monovalent Fab antibody fragments neutralized chlamydial infectivity for both HaK and HeLa cells. Immunofluorescence analysis of HeLa 229 cells with a panel of monoclonal antibodies specific to human Fc gamma receptors revealed the expression of cell surface Fc gamma RIII. We propose that Fc gamma RIII may obscure the chlamydia-neutralizing activity of certain IgG isotypes by facilitating the Fc gamma R-mediated entry of chlamydiae into HeLa 229 cells. These findings may help explain the inconsistencies that are commonly observed in results when HeLa 229 cells are used in chlamydia neutralization assays.
Deletion of the BCSP31 gene of Brucella abortus by replacement
Authors: Halling S M , Detilleux P G , Tatum F M , Judge B A , Mayfield J E
Published: January 2020

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.
DNA sequence and in vitro mutagenesis of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans
Authors: Duncan M J , Hillman J D
Published: January 2020

Previously, the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase gene of Streptococcus mutans JH1000 was cloned into Escherichia coli (J. D. Hillman, M. J. Duncan, and K. P. Stashenko, Infect. Immun. 58:1290-1295, 1990). In the present study, the nucleotide sequence of 1.29 kb of S. mutans DNA which contained the promoter and protein-coding region of the gene was determined. In vitro disruption of the gene was achieved by deletion of the promoter and a major portion of the protein-coding sequence. Subsequently, a tetracycline resistance gene from S. mutans was inserted at the deletion site as a marker for selection. In addition, evidence from Southern hybridization showed that S. mutans JH1000 contained a single copy of the lactate dehydrogenase gene.
Identification and purification of transferrin- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica
Authors: Menozzi F D , Gantiez C , Locht C
Published: January 2020

Bordetella pertussis and Bordetella bronchiseptica were both able to grow in iron-deficient medium when supplemented with iron-saturated human lactoferrin or transferrin but not with human apotransferrin. Direct contact between the transferrins and the Bordetella cells did not appear to be required for growth but considerably improved the growth of the organisms. Analysis of B. pertussis and B. bronchiseptica whole-cell lysates from cultures carried out in iron-deficient or iron-replete media revealed iron-repressible proteins (IRPs) of 27 kDa in B. pertussis and of 30, 32, 73.5, and 79.5 kDa in B. bronchiseptica. Iron-inducible proteins of 16, 23.5, 36.5, and 92.5 kDa and of 17, 23.5, 70, 84, and 91 kDa were also identified in B. pertussis and B. bronchiseptica, respectively. By use of affinity chromatography with iron-saturated human lactoferrin or transferrin as ligands, the 27- and 32-kDa IRPs from B. pertussis and B. bronchiseptica, respectively, were specifically isolated. By using iron-chelated affinity columns, we showed that these proteins exhibit an affinity for iron. Cell fractionation experiments indicated that both of these proteins are probably associated with the outer membrane. Growth of the organisms under modulating conditions showed that the production of these IRPs is not under the genetic transcriptional control of vir or bvg, the general virulence regulon in Bordetella spp.
ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S
Authors: Coburn J , Gill D M
Published: January 2020

Pseudomonas aeruginosa exoenzyme S ADP-ribosylates p21ras and several related proteins. ADP-ribosylation of p21ras does not alter interactions with guanine nucleotides. The ras-related GTP-binding proteins, including Rab3, Rab4, Ral, Rap1A, and Rap2, are also substrates; given these results, we propose a model for the role of exoenzyme S in pathogenesis.
Testosterone-induced abrogation of self-healing of Plasmodium chabaudi malaria in B10 mice: mediation by spleen cells
Authors: Benten W P , Bettenhaeuser U , Wunderlich F , Van Vliet E , Mossmann H
Published: January 2020

This study investigates the suppressive effect of testosterone (Te) on the self-healing of Plasmodium chabaudi malaria in female mice of the strain C57BL/10, and, in particular, the possible role of spleen cells in mediating this Te effect. Our data show the following. (i) About 80% of B10 mice infected with 10(6) P. chabaudi-infected erythrocytes are capable of self-healing the infections. This capability is progressively impaired and finally abrogated after pretreating the B10 mice with Te for 3 weeks. (ii) The spleen is Te responsive. This becomes evident in a reduction of total spleen cells from 1.05 x 10(8) to 0.54 x 10(8) on average after Te treatment for 3 weeks. Moreover, Te treatment causes an increase in the relative proportion of CD8+ cells by about 4% and a decrease of Ig+ cells by about 4.5%, as revealed by flow cytometry. (iii) Spleen cells mediate the suppressive Te effect as revealed by adoptive transfer experiments. The percentage of self-healing mice dramatically decreases to about 8% when they receive, just prior to infection, nucleated spleen cells isolated from mice treated with Te for 3 weeks. This suppressive effect can be transferred by T cells in particular but also by non-T cells, though to a lesser extent. (iv) The adoptively transferred cells mediate their suppressive effect on self-healing only if the recipient mice receive Te during infection. Our data suggest that spleen cells become functionally changed by the Te treatment for 3 weeks. Particularly T cells, but also non-T cells, gain P. chabaudi-specific suppressive activities, and the cells require a Te-induced factor(s) to mediate these activities.
Continuous infusion of Escherichia coli endotoxin in vivo primes in vitro superoxide anion release in rat polymorphonuclear leukocytes and Kupffer cells in a time-dependent manner
Authors: Mayer A M , Spitzer J A
Published: January 2020

Continuous infusion of a nonlethal dose of Escherichia coli lipopolysaccharide (LPS) (0.5 mg/kg) induced early (3 h) accumulation of polymorphonuclear leukocytes (PMNL) in rat liver followed by later (30 h) greater extravasation of mononuclear phagocytes (MNP) (E. B. Rodriguez de Turco and J. A. Spitzer, J. Leukocyte Biol. 48:488-494, 1990). Nonparenchymal liver cells from rats treated for 3 and 30 h with LPS were recovered by centrifugal elutriation, yielding a 23-ml/min fraction (endothelial cells) and a 45-ml/min fraction (PMNL, Kupffer cells, and MNP), and compared for their capacity for basal and agonist-stimulated superoxide (O2-) production. Stimulation with phorbol myristate acetate and opsonized zymosan caused a dose-dependent release of O2- from the 45-ml/min fraction derived from rats treated for 3 h with saline, but not from the 23-ml/min fraction. Further purification of the 45-ml/min fraction by discontinuous density gradient centrifugation into a Kupffer and a PMNL fraction revealed that most of the agonist-induced O2- release was generated by infiltrating PMNL at this early time point of LPS infusion. By 30 h of LPS infusion, although enhancement of the phorbol-12-myristate-13-acetate- and opsonized zymosan-stimulated release of O2- was observed in the 45-ml/min fraction, but not in the 23-ml/min fraction, the maximum release of O2- was smaller than that observed in the rats treated for 3 h. Our results support the following conclusions: (i) after a 3-h LPS infusion, PMNL found in the liver in increased numbers are also highly primed for agonist-stimulated release of O2-, while Kupffer cell priming is of a lesser extent; (ii) after a 30-h infusion of LPS, infiltrating MNP found in the liver in increased numbers are primed for agonist-induced O2- release, while priming of PMNL has diminished; (iii) at both 3 and 30 h of LPS infusion, liver endothelial cells are not significantly primed for agonist-stimulated O2- release; and (iv) in vivo priming by LPS infusion at both 3 and 30 h was not reversed by the experimental method used for cell recovery (ca. 3 h), thus suggesting that in vivo LPS priming of O2- release may ultimately lead to severe impairment of liver function and metabolism observed during endotoxemia and sepsis if not therapeutically blocked at an early time point.
Isolation and sequence analysis of the Chlamydia pneumoniae GroE operon
Authors: Kikuta L C , Puolakkainen M , Kuo C C , Campbell L A
Published: January 2020

Chlamydia pneumoniae has emerged as an important human respiratory pathogen. From a lambda gt11 gene bank constructed from C. pneumoniae isolate AR-39 DNA, an immunoreactive plaque containing a 3.0-kb insert was purified. In immunoblots, a 60-kDa protein was recognized by anti-C. pneumoniae rabbit immune serum. The recombinant protein was reactive with a Chlamydia genus-specific monoclonal antibody recognizing a 60-kDa protein found in the Sarkosyl-soluble fraction and with rabbit immune serum prepared against the Chlamydia trachomatis 60-kDa GroEL homolog associated with the delayed-type hypersensitivity response. DNA sequence analysis confirmed that the C. pneumoniae gene product is an analog of the C. trachomatis delayed-type hypersensitivity antigen and the Escherichia coli GroEL heat shock protein.
Interleukin-1 administration to C3H/HeJ mice after but not prior to infection increases resistance to Salmonella typhimurium
Authors: Morrissey P J , Charrier K
Published: January 2020

Interleukin-1 (IL-1) treatment of C3H/HeN and C3H/HeJ mice prior to infection with Salmonella typhimurium increased the survival fraction only in C3H/HeN mice. IL-1 administration after infection resulted in a significant increase in mean survival time in C3H/HeJ but not C3H/HeN mice. Bacterial growth in IL-1-treated C3H/HeJ mice was less than that in control mice.
Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron
Authors: Tompkins G R , O'Neill M M , Cafarella T G , Germaine G R
Published: January 2020

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.
Molecular cloning and analysis of recombinant major antigens of Ehrlichia risticii
Authors: Dutta S K , Shankarappa B , Mattingly-Napier B L
Published: January 2020

The genome of Ehrlichia risticii, the etiologic agent of Potomac horse fever, was cloned in the lambda gt11 expression vector. The efficiency of recombinant phage production with different restriction fragments of E. risticii DNA was generally between 20 and 95%. The antigen-positive frequency, detected by immunoscreening with E. risticii antibodies, was between 8 and 40 per 10(4) recombinants. Four (70, 55, 51, and 44 kDa) major antigens of E. risticii were identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. Characterization of three (70, 55, and 44 kDa) of these recombinant antigens indicated that the 70- and 44-kDa polypeptides were beta-galactosidase fusion products that were dependent on isopropylthiogalactoside induction for expression; they contained about 50 and 73%, respectively, of the native polypeptides. The 55-kDa antigen was a nonfusion protein expressed independently of isopropylthiogalactoside induction; it was a complete protein with a molecular weight identical to that of its native counterpart. The cloned E. risticii DNAs from of the recombinants expressing 70-, 55-, and 44-kDa proteins were 3.5, 3.9, and 4.8 kb, respectively, in size, and they were unique. The insert DNAs hybridized to multiple restriction fragments of the genomic DNA, the sum of the sizes of which was much greater than that of the corresponding insert. Mice immunized with the affinity-purified 55-kDa recombinant antigen produced a high titer of antibody in serum as measured by an enzyme-linked immunosorbent assay and gave a monospecific reaction by Western immunoblotting. Challenge infection of these immunized mice showed low protection from clinical infection.
Palmitoylated proteins in Ureaplasma urealyticum
Authors: Thirkell D , Myles A D , Russell W C
Published: January 2020

After incubation of Ureaplasma urealyticum serotype 8 in the presence of 3H-labeled palmitic acid, about 25 acylated proteins were detected by electrophoresis and fluorography. Of these, at least six were shown to be antigenic by immunoprecipitation of solubilized palmitate-labeled cells with a homologous polyclonal serum. These six included the serotype 8-specific surface-expressed 96-kDa antigen. After phase partition of palmitate-labeled cells with Triton X-114, all but six acylated proteins partitioned entirely into the detergent phase. The others, including the 96-kDa antigen, partitioned preferentially into the detergent phase and were apparently amphipathic. These results are consistent with the acylated proteins being mainly membrane associated.
Purification and characterization of a protective antigen from Eimeria tenella
Authors: Karkhanis Y D , Nollstadt K A , Bhogal B S , Ravino O , Pellegrino R , Crane M S , Murray P K , Turner M J
Published: January 2020

Chickens were partially protected against coccidiosis induced by Eimeria tenella by using extracts prepared from sonicated sporulated oocysts or from sonicated sporozoites. Following gel filtration of either extract, most of the protective antigens were confined to a single pool of proteins in the molecular mass range between 20 and 30 kDa. Further purification of proteins from the protective pool of sporulated oocyst extract yielded a protective polypeptide with a molecular mass of 26 kDa. An antiserum raised to this pool identified a polypeptide with a molecular mass of 22 kDa in the protective pool from a similarly prepared extract of sporozoites of E. tenella. In subsequent studies, this antiserum was used as an aid in cloning protective polypeptides.
Cross-protection against four species of chicken coccidia with a single recombinant antigen
Authors: Crane M S , Goggin B , Pellegrino R M , Ravino O J , Lange C , Karkhanis Y D , Kirk K E , Chakraborty P R
Published: January 2020

A cDNA clone, SO7', from an Eimeria tenella cDNA library was inserted into the high-expression vector pJC264 and was expressed in Escherichia coli as a fusion protein, CheY-SO7', with a molecular mass of approximately 36 kDa. By using the purified recombinant antigen to immunize young chicks, it was demonstrated that a single dose, without adjuvant, not only protected against severe coccidiosis induced by infection with E. tenella but also protected chicks challenged with the heterologous species Eimeria acervulina, E. maxima, and E. necatrix. By using rabbit antiserum raised against recombinant CheY-SO7', Western blot (immunoblot) analysis of sporulated oocysts of all seven major species of chicken coccidia showed that all species tested contained proteins characteristic of the B class of antigens, of which CheY-SO7' is representative. It seems likely that a single B antigen could protect chickens against severe coccidiosis caused by infection with any of these Eimeria species. Although chicks exposed to prolonged, natural infection develop antibodies to B antigen, active immunization of young chicks with a protective dose of CheY-SO7' does not elicit a humoral antibody response, suggesting that the partial protection results from cell-mediated effector mechanisms. In addition, the cross-protective nature of the immunity indicates that the response to B antigen is different from that induced by natural infection, which elicits a species-specific immunity. To date, the protection induced by B antigen immunization, although remarkable for a single recombinant protein, is not sufficient to compete with prophylactic chemotherapy.
Evidence that a 16-kilodalton integral membrane protein antigen from Schistosoma japonicum adult worms is a type A2 phospholipase
Authors: Rogers M V , Henkle K J , Herrmann V , McLaren D J , Mitchell G F
Published: January 2020

Type A2 phospholipase (PLA2) activity has been observed in integral membrane protein extracts of Schistosoma japonicum. Antiserum raised against bee venom PLA2 recognized a single 16-kDa band in the parasite extracts; it also localized to antigen in the gut lining of fixed adult schistosomes as shown by immunofluorescence techniques. Evidence was obtained that the molecule was expressed at low levels in comparison with other integral membrane proteins and was weakly immunogenic in rabbits. Two oligonucleotide probes were constructed on the basis of highly conserved regions between the nucleotide sequences of rat, bovine, rattlesnake, and bee venom PLA2; these probes were used to isolate S. japonicum genomic DNA phage clones. A 1.8-kb FnuD2 fragment was shown by Southern blot analysis to strongly hybridize with the 5' 32P-labeled PLA2 oligonucleotides in both S. japonicum genomic DNA and DNA from one of the phage clones. The nucleotide and predicted amino acid sequences of this fragment revealed homology with the C terminus of PLA2s from different species.
Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice
Authors: Lu Y S , Aguila H N , Lai W C , Pakes S P
Published: January 2020

The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 10(6) CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P less than 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P less than 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P greater than 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs.
Gamma interferon suppresses acute and chronic Trypanosoma cruzi infection in cyclosporin-treated mice
Authors: McCabe R , Meagher S , Mullins B
Published: January 2020

To determine if exogenous gamma interferon is effective in immunosuppressed mice infected with Trypanosoma cruzi, recombinant murine gamma interferon was administered to cyclosporin-treated mice with either acute or chronic T. cruzi infection. Gamma interferon significantly decreased parasitemia and prevented death in acutely infected mice. Parasitemias and mortality of mice treated with both gamma interferon and cyclosporin were similar to those of immunocompetent controls. In chronically infected mice, cyclosporin treatment produced significantly more organ explant cultures positive for T. cruzi. Fewer positive cultures, particularly for spleen and heart, were obtained from cyclosporin-treated mice when they also received gamma interferon. Ketoconazole treatment of mice resulted in no positive cultures. Cyclosporin treatment did not prevent activation of peritoneal macrophages by parenteral gamma interferon, nor did it have a consistent effect on serum titers of alpha/beta or gamma interferon in response to a second challenge inoculum of T. cruzi. These data indicate that exogenous gamma interferon suppresses acute and chronic T. cruzi infection in cyclosporin-treated mice but that gamma interferon is not as effective as the relatively specific antimicrobial ketoconazole. Gamma interferon activates macrophages despite cyclosporin treatment, and its effects appear to be tissue specific.
Acquired resistance to Giardia muris in X-linked immunodeficient mice
Authors: Skea D L , Underdown B J
Published: January 2020

A previous study from this laboratory (D. P. Snider, D. Skea, and B. J. Underdown, Infect. Immun. 56:2838-2842, 1988) indicated that immunodeficient mice expressing the xid gene develop prolonged infections with Giardia muris, unlike immunocompetent mice, which eliminate the intestinal protozoan parasite in 8 to 10 weeks. In this study, CBA/N (xid) and CBA/Ca mice were infected with G. muris cysts and at various times following this primary infection were cured by treatment with metronidazole. In contrast to the marked differences in the ability of xid and normal mice to eliminate a primary infection, mice of both strains were resistant to a secondary challenge of G. muris cysts. These data imply that the mechanism(s) responsible for elimination of a primary infection is not identical to those required to resist a secondary challenge infection. Splenocytes from immunocompetent CBA/Ca mice (but not immunodeficient CBA/N mice) could transfer the ability to eliminate a primary G. muris infection to irradiated mice of either strain. In contrast, splenocytes from previously infected CBA/Ca mice could not transfer resistance to a challenge infection, further supporting the hypothesis that there are differences between mechanisms required to eliminate a primary infection and those necessary to resist a second challenge infection.
Recombinant murine beta interferon enhances resistance of mice to systemic Mycobacterium avium infection
Authors: Denis M
Published: January 2020

Susceptible BALB/c mice were infected with Mycobacterium avium TMC 702. Groups of mice were then infused with 10(4) U (approximately 400 U/h) of murine beta interferon (IFN-beta) via a minipump system, and the progression of the infection was assessed. Mice infused with IFN-beta showed superior resistance to infection, as determined by reduced bacterial growth in the livers and spleens of infected animals, (1-log reduction in bacterial CFU at 2 months postinfection; P less than 0.001). This was corroborated by the fact that resident peritoneal macrophages treated with IFN-beta in vitro (10(2) U/ml) were more bacteriostatic for M. avium TMC 702 than their untreated counterparts. Overall, these findings suggest an important role for IFN-beta in mycobacterial infections.
Enzymatically deacylated Neisseria lipopolysaccharide (LPS) inhibits murine splenocyte mitogenesis induced by LPS
Authors: Erwin A L , Mandrell R E , Munford R S
Published: January 2020

Acyloxyacyl hydrolase is a leukocyte enzyme that selectively removes the secondary acyl chains from the lipid A moiety of gram-negative bacterial lipopolysaccharides (LPS). As predicted by the reported contribution of secondary acyl chains to the bioactivities of lipid A analogs, enzymatic deacylation of Salmonella typhimurium Rc LPS substantially reduces its potency in the dermal Shwartzman reaction and in several in vitro assays that measure responses of human endothelial cells and neutrophils, whereas the potency of this LPS for inducing murine splenocyte mitogenesis is affected much less. In the experiments described here, we studied the impact of acyloxyacyl hydrolysis on the bioactivities of several LPS that differ from Salmonella LPS in carbohydrate and lipid A structures. Deacylated LPS from Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, and S. typhimurium were similarly reduced in potency in the Limulus lysate test (30- to 60-fold reduction in potency relative to the corresponding mock-treated LPS), and the ability of all of these deacylated LPS to stimulate neutrophil adherence to human endothelial cells was reduced by a factor of 100 or more. For LPS from E. coli, H. influenzae, and Pseudomonas aeruginosa, the impact of deacylation on spleen cell mitogenesis was also similar to that observed for S. typhimurium LPS: deacylation reduced potency by less than 15-fold. Unexpectedly, the potency of Neisseria LPS in the murine splenocyte mitogenicity test was reduced over 100-fold by deacylation, and deacylated Neisseria LPS could block the mitogenic activity of Neisseria and Salmonella LPS. These studies indicate that the contribution of secondary acyl chains to the bioactivities of a given LPS cannot be predicted with confidence from the reported structure-activity relationships of lipid A or from the behavior of other deacylated LPS.
Proteins released from Mycobacterium tuberculosis during growth
Authors: Andersen P , Askgaard D , Ljungqvist L , Bennedsen J , Heron I
Published: January 2020

Proteins secreted from Mycobacterium tuberculosis during growth are believed to be important for protective immunity against tuberculosis. We have investigated the growth of M. tuberculosis in an enriched liquid medium. The release of isocitrate dehydrogenase from the bacilli served as a marker of autolysis and was observed during the late logarithmic growth phase. The release of proteins during the culture period was investigated by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three major groups of proteins, which differed markedly with respect to profile of release and location in intact bacilli, were defined. A short-term filtrate devoid of autolytic products was defined and found to be composed of 33 major components. Five proteins were identified by monoclonal antibodies. Pronounced superoxide dismutase activity was detected in the filtrate. The enzyme was purified and identified as a dominating component of short-term filtrate.
Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells
Authors: Mobley H L , Chippendale G R , Swihart K G , Welch R A
Published: January 2020

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 10(6) CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P less than 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available.