bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets
Authors: Eaton K A , Brooks C L , Morgan D R , Krakowka S
Published: January 2020

A mutant strain of Helicobacter pylori with weak urease activity was created by using N-methyl-N'-nitro-N-nitrosoguanidine. The urease activity of the mutant (0.036 +/- 0.009 nmol of urea per micrograms of bacterial protein per min) was 0.4% of that of the parental strain (8.20 +/- 2.30 nmol of urea per micrograms of bacterial protein per min). The mutant was otherwise indistinguishable from the parental strain. Both demonstrated prominent catalase and oxidase activities, and both produced vacuolating cytotoxin. Restriction endonuclease and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and ultrastructure were identical for the two strains. The mutant was fully motile, as evaluated by spreading in soft agar and by direct microscopic examination. Growth rate and colony size and morphology were identical for the mutant and parental strains. Seventeen gnotobiotic piglets were challenged with either the mutant or the parental strain and sacrificed 3 or 21 days after challenge. Gastric tissue was examined histologically and cultured for H. pylori. Of seven piglets challenged with the parental strain, all became infected. H. pylori was not recovered from any of 10 piglets challenged with the urease-negative strain. Lymphofollicular gastritis was present in all seven piglets challenged with the parental strain but in none of the piglets challenged with the urease-negative strain. These results suggest that prominent urease activity is essential for colonization by H. pylori.
Borreliacidal activity of sera from hamsters infected with the Lyme disease spirochete
Authors: Lovrich S D , Callister S M , Schmitz J L , Alder J D , Schell R F
Published: January 2020

An in vitro borreliacidal assay that accurately reflects the levels of protective antibody determined by passive transfer of immunity studies was developed. Borreliacidal antibody in sera obtained from normal hamsters infected with Borrelia burgdorferi was readily detected. When immune serum containing complement was incubated with B. burgdorferi organisms, spirochetes were killed within 2 h. Treating immune serum with anti-hamster immunoglobulin G abrogated the borreliacidal activity. Killing of B. burgdorferi in serum was detected 1 week after infection; it peaked at week 3 and gradually declined. Relatively high levels of borreliacidal antibody were found, especially in week 3 immune serum, which could be diluted 1,280-fold. The decrease in borreliacidal antibody after infection may account for occurrences of reinfection and the remitting course of Lyme disease.
Pseudomonas aeruginosa exoenzyme S is an adhesion
Authors: Baker N R , Minor V , Deal C , Shahrabadi M S , Simpson D A , Woods D E
Published: January 2020

Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.
Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella typhi are deficient for entry into mammalian cells
Authors: Galán J E , Curtiss R
Published: January 2020

Invasion of intestinal epithelial cells is an essential virulence factor of salmonellae. A group of genes, invABC and invD, that allow Salmonella typhimurium to penetrate cultured epithelial cells have previously been characterized (J. E. Galán and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). The distribution of these genes among Salmonella isolates belonging to 37 different species or serovars was investigated by Southern and colony blot hybridization analyses. Regions of high sequence similarity to the invABC genes were present in all Salonella isolates examined, while regions of sequence similarity to the invD gene were present in all but one (S. arizonae) of the isolates tested, with little restriction fragment length polymorphism. Sequences similar to these genes were not detected in strains of Escherichia coli, Yersinia spp., or Shigella spp. invA mutants (unable to express the invABC genes) of several Salmonella species or serovars, including S. typhi, were constructed and examined for their ability to penetrate Henle-407 cells. All mutants were deficient for entry into cultured epithelial cells, indicating that the invABC genes were not only present in these strains but also functional.
Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription
Authors: Carlsson A , Engström P , Palva E T , Bennich H
Published: January 2020

Attacins are antibacterial proteins synthesized by pupae of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. In this report we show that the previously described, attacin-induced alteration in the structure and the permeability of the outer membrane of Escherichia coli is associated with a specific inhibition of the synthesis of several outer membrane proteins, including OmpC, OmpF, OmpA, and LamB. The inhibition is expressed as a reduction in the steady-state mRNA levels and is at least in part the results of a block in transcription of the corresponding genes. Transcription directed by the promoter of ompR, the positive regulator of ompC and ompF expression in response to environmental conditions, is also affected by attacin. The effects on mutant strains show that the primary activity of attacin is not mediated by the ompR-envZ regulatory system. Instead our data suggest the existence in E. coli of a previously unknown system for the transcriptional regulation of a large set of outer membrane proteins previously not known to be coordinately regulated. We propose that the activity of attacin is directed towards this system.
The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene
Authors: Green B A , Farley J E , Quinn-Dey T , Deich R A , Zlotnick G W
Published: January 2020

Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae.
Isolation of an intracellular bacterium from hamsters (Mesocricetus auratus) with proliferative ileitis and reproduction of the disease with a pure culture
Authors: Stills H F
Published: January 2020

An intracellular bacterium was isolated from hamsters (Mesocricetus auratus) with proliferative ileitis. The organism was isolated in Intestine 407 and GPC-16 cell cultures (incubated in a microaerophilic atmosphere) from isolated and lysed epithelial cells from hamsters with proliferative ileitis. The bacterium measured 1.4 to 1.7 microns in length by 0.26 to 0.34 microns in width, was slightly curved, and had an irregular trilaminar cell wall. Inoculation of hamsters with a cell culture lysate containing the organism or a 0.65-microns-pore-size filtrate of an infected-cell lysate resulted in the typical lesions of proliferative ileitis in approximately 50% of the animals in 28 days. Hamsters inoculated with uninfected cells or a 0.2-microns-pore-size filtrate of an infected-cell lysate remained uninfected. Attempts to propagate the organism on cell-free media have been unsuccessful.
Identification of larval cross-reactive and egg-specific antigens involved in granuloma formation in murine schistosomiasis mansoni
Authors: Lukacs N W , Boros D L
Published: January 2020

Cross-reactive humoral immune responses between antigens of different developmental stages of the worm Schistosoma mansoni have previously been demonstrated. In contrast, information on antigenic cross-reactivity at the T-cell level is still very sparse. The present study examined the cross-reactive T-cell responses to eggs and crude and fractionated soluble egg antigens (SEA) in infected mice prior to (from 0 to 4 weeks of infection) and after (5 weeks and onwards) egg deposition. Splenic lymphocyte proliferation to unfractionated SEA was detected as early as 2 weeks postinfection and increased rapidly by 4 weeks postinfection. Injections of live eggs into the lungs of infected mice at 4 weeks postinfection demonstrated enhanced granuloma formation, indicating the presence of primed T cells that respond to egg antigens. Further experiments with the artificial granuloma model and polyacrylamide gel electrophoresis-separated SEA fractions demonstrated that in mice infected for 4 weeks the 60- to 66-, 93- to 125-, and greater than 200-kDa SEA fraction-coated beads elicited significant pulmonary granulomas. By 6 weeks postinfection, when eggs are deposited in the livers, in addition to the cross-reactive fractions (60 to 66, 93 to 125, and greater than 200 kDa), beads coated with fractions of 25 to 30, 32 to 38, and 70 to 90 kDa also elicited significant granulomatous reactions. These antigenic fractions are considered to have elicited egg stage-specific T-cell responsiveness. In addition hepatic granuloma T cells from the 6th week of infection demonstrated the strongest blastogenic response to the 60- to 66-kDa cross-reactive fraction. Thus, in vitro and in vivo experiments demonstrated T-cell cross-reactivity between the larval and egg stages of the worm. On the basis of these observations, the appearance of the primary circumovum granulomatous response in infected mice is considered to represent the sum of larval cross-reactive and egg-specific T-cell responsiveness.
Growth of Francisella spp. in rodent macrophages
Authors: Anthony L D , Burke R D , Nano F E
Published: January 2020

We examined the nature of the interactions between the facultative intracellular pathogens Francisella tularensis and F. novicida and rodent macrophages. Growth of F. tularensis LVS was observed in macrophage monolayers from mice, guinea pigs, or rats. In contrast, F. novicida grew in macrophages from mice and guinea pigs but not in macrophages from rats. Transmission electron microscopy studies indicated that both Francisella species survive within macrophage phagosomes that are unfused with lysosomes.
Effects of Passive Antibody on Early Pathogenesis of Marek's Disease
Authors: Calnek B. W.
Published: January 2020

The effects of passive Marek's disease virus (MDV) antibody were characterized in three experiments in which 1- or 2-day-old PDRC chickens were inoculated with cell-associated MDV. Antibody acquired naturally from the dam, or injected prior to, or at the time of, virus inoculation, failed to prevent infection, but the incidence and severity of the acute necrotizing disease were reduced. Also, the number of tissues with viral antigen (fluorescent antibody test) and the amount of antigen in positive tissues was lowered, and fewer infected cells in spleen (in vitro assay) were detected in antibody-positive chicks. Serum containing MDV antibody was efficacious when injected prior to infection and had some protective effect when first given at day 4 but not at day 7 after virus inoculation. Normal antibody-free serum was ineffective, and chickens with naturally acquired (maternal) antibody against turkey herpesvirus (a virus antigenically related to MDV) were not protected against MDV infection.
Comparison of the Virion Polypeptides of Group B Arboviruses
Authors: Shapiro Daniel , Trent Dennis , Brandt Walter E. , Russell Philip K.
Published: January 2020

The virion polypeptides of eight group B arboviruses were compared by polyacrylamide gel coelectrophoresis. All contained three polypeptides, V-1, V-2, and V-3. The mosquito-borne viruses had electrophoretically similar small membrane polypeptides (V-1); the tick-borne viruses had a V-1 of significantly decreased mobility.
Effect of
Corynebacterium acnes
on Interferon Production in Mice
Authors: Farber P. A. , Glasgow L. A.
Published: January 2020

Exposure to
Corynebacterium acnes
, the most prominent member of our normal skin flora, produces stimulation of lymphoid tissue and certain reticuloendothelial system functions, as well as the immune response. Alteration of the host response is extended by these studies to include changes in the pattern of interferon production in response to a representative group of inducing agents. Serum interferon levels induced by the injection of endotoxin in mice are enhanced, whereas interferon production after injection of Newcastle disease virus, Chikungunya virus, and polyinosinic:polycytidylic acid is depressed in animals inoculated with viable or nonviable
C. acnes
organisms.
Isolation of Porcine Immunoglobulins and Determination of the Immunoglobulin Classes of Transmissible Gastroenteritis Viral Antibodies
Authors: Saif Linda J. , Bohl Edward H. , Gupta R. K. Paul
Published: January 2020

The porcine immunoglobulins M (IgM), A (IgA), and G (IgG) were isolated and purified and some of the properties of the porcine milk IgA were examined. Monospecific antisera which were prepared against these immunoglobulins in rabbits were then used to absorb a particular class of immunoglobulin from sow serum, colostrum, and milk in an attempt to identify the immunoglobulin classes of neutralizing antibodies to the porcine enteric virus, transmissible gastroenteritis (TGE). The results of these absorption studies suggest that in colostrum and milk from sows experimentally (orally) or naturally infected with live virulent TGE virus, IgA is the predominant immunoglobulin class of TGE antibodies. Both IgA and IgG TGE antibodies appeared to be present in the serum from these sows, but with IgG TGE antibodies predominating. In contrast, in the serum, colostrum and milk from sows vaccinated intramuscularly or intramammarily with live attenuated TGE virus, the TGE antibody activity was associated mainly with the IgG class of immunoglobulins. These results provide additional data indicating that the route of infection or vaccination markedly influences the immunoglobulin class of antibodies in colostrum and milk. Secondly, IgA antibodies in mammary secretions are probably essential for providing optimal passive immunity of nursing pigs against infection with TGE virus.
Classification of
Neisseria meningitidis
Group B into Distinct Serotypes IV. Preliminary Chemical Studies on the Nature of the Serotype Antigen
Authors: Frasch Carl E. , Chapman S. Stephen
Published: January 2020

Group B
Neisseria meningitidis
has been subdivided into 11 distinct serotypes by a sensitive bactericidal inhibition technique. The antigens responsible for induction of bactericidal type-specific antibodies were found to be extractable from the group B cells with heating at 100 C either by 0.017
n
HCl in saline or by normal saline. These extracted serotype antigens were detected by a capillary precipitin test. The development of methods for extraction and assay of the serotype antigens permitted studies on their immunochemistry. The serotype antigens were distinct from the group-specific substance. Acid extracts contained abundant serotype antigen, but were devoid of group-specific substance. The identity of serotype antigens as proteins was confirmed by their sensitivity to Pronase and trypsin. The molecular weight of these antigens as estimated by G-200 Sephadex chromatography and by electrophoresis in polyacrylamide gels is in excess of 200,000 daltons. Saline extracts containing the serotype antigen could be fractionated into three distinct fractions with acetic acid:
p
H 4.5 and
p
H 3.5 precipitated fractions and a
p
H 3.5 supernatant fraction. The
p
H 4.5 precipitated fraction contained the serotype antigen.
Titration of Antibodies to Salmonella O Antigens by Enzyme-Linked Immunosorbent Assay
Authors: Carlsson Hans E. , Lindberg Alf A. , Hammarström Sten
Published: January 2020

A new type of immunoassay (enzyme-linked immunosorbent assay, ELISA) has been used for the detection of antibodies against
Salmonella
O antigens. The results show that ELISA is specific and highly sensitive. When compared with the Widal reaction, passive hemagglutination, and quantitative precipitation, ELISA was shown to be more sensitive. In addition, immunoglobulin G and immunoglobulin M antibodies were both detected approximately as efficiently with ELISA.
Plaque Formation in Tissue Cultures by
Rickettsia rickettsi
Isolated Directly from Whole Blood and Tick Hemolymph
Authors: Wike David A. , Burgdorfer Willy
Published: January 2020

A simple technique is described for isolation of
Rickettsia rickettsi
directly from tick hemolymph and whole blood of rickettsemic guinea pigs by means of the plaque assay technique in primary chicken embryo tissue cultures. Plaque-forming units per drop of hemolymph were almost 100-fold greater for partially engorged ticks than for unengorged ticks. Rickettsemia in guinea pigs fed upon by infected ticks was detected as early as 24 hr before fever. No morphological differences were noted between plaques formed by rickettsiae isolated from tick hemolymph or from whole guinea pig blood.
Abortive Infection of Human Diploid Cells by Murine Cytomegalovirus
Authors: Kim Kwang Soo , Carp Richard I.
Published: January 2020

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 μg/ml) or puromycin (20 μg/ml), but 5′-fluoro-2′-deoxyuridine at 1 × 10
−4
m
did not prevent CPE or the development of intranuclear inclusions.
Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli
Authors: Hunter S E , Clarke I N , Kelly D C , Titball R W
Published: January 2020

The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.
In vitro characterization of T cells from Mycobacterium w-vaccinated mice
Authors: Singh I G , Mukherjee R , Talwar G P , Kaufmann S H
Published: January 2020

Tuberculosis caused by the intracellular bacterial pathogen Mycobacterium tuberculosis still represents a major health problem, and its effective control would best be accomplished by active vaccination. Although vaccination with M. bovis BCG has proven highly effective in certain parts of the world, in several developing countries it has been found to confer only marginal protection. Hence, novel vaccination strategies are warranted. Mycobacterium w is a saprophytic cultivable mycobacterium which shares several antigens with M. tuberculosis. In the murine system, vaccination with killed M. w was found to protect against subsequent tuberculosis. In order to characterize the responsible immune mechanisms more precisely, mice were vaccinated with killed M. w and T cells restimulated in vitro with mycobacterial antigens. These T cells produced interleukin 2 and gamma interferon but no detectable interleukin 4 and interleukin 5. Killed M. w induced significantly stronger T-cell responses than killed M. tuberculosis, and both vaccination regimes were markedly improved by administration in a mild adjuvant, i.e., the Ribi adjuvant containing trehalose dimycolate, monophosphoryl lipid A, and mycobacterial cell wall skeleton. Our data suggest that M. w-induced immunity against M. tuberculosis rests primarily on TH1 cells, which are thought to be of major relevance for acquired antituberculosis resistance. Our study therefore provides a further step toward the identification of a novel tuberculosis vaccine.
Cell-free system responsible for internal radiolabeling of glycopeptidolipids of the Mycobacterium avium complex
Authors: Ramasesh N , Wright E L , Barrow W W
Published: January 2020

Internal radiolabeling of serotype-specific glycopeptidolipids with [14C]mannose was accomplished with a cell-free system derived from serotype 20 of the Mycobacterium avium complex. Similar radiolabeling was not apparent with a cell-free system derived from the rough colony variant, previously shown to be devoid of glycopeptidolipids. Although a comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis of the parent and rough variant strains revealed a close similarity, there were some proteins unique to the parent strain.
Adherence of oral streptococci to salivary glycoproteins
Authors: Murray P A , Prakobphol A , Lee T , Hoover C I , Fisher S J
Published: January 2020

We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity.
Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis
Authors: Connell T D , Holmes R K
Published: January 2020

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.
Characterization of I/F1 glycoprotein as a receptor for Mycoplasma pneumoniae
Authors: Hengge U R , Kirschfink M , König A L , Nicklas W , Roelcke D
Published: January 2020

Serologic evidence of anti-I and anti-Fl cold agglutinins occurring in mycoplasma infections led to the isolation of I/Fl glycoprotein from human erythrocyte membranes. Mycoplasma pneumoniae bound to purified I/Fl glycoprotein in a dose-dependent fashion depending on sialylated carbohydrate determinants. This was shown by the decreased binding of mycoplasmas to either sialidase-treated I/Fl glycoprotein (dot blot analysis) or sialidase-treated erythrocytes (hemagglutination test). Structural properties of the receptor for optimal binding could be explored by hemagglutination inhibition assays. Glycophorins were excluded as receptors. These results indicate that Fl (and I) antigens are receptors for M. pneumoniae.
Influence of Actinobacillus pleuropneumoniae serotype 2 and its cytolysins on porcine neutrophil chemiluminescence
Authors: Dom P , Haesebrouck F , Kamp E M , Smits M A
Published: January 2020

The effects of Actinobacillus pleuropneumoniae serotype 2 and its metabolites on the oxidative activity of porcine neutrophils were studied by using a chemiluminescence technique. Viable A. pleuropneumoniae stimulated the production of oxygen radicals by neutrophils. After having reached a peak value, the oxidative activity decreased until a total inhibition of the oxidative activity of the neutrophils was achieved. All effects were neutralized with homologous convalescent-phase pig sera which had been adsorbed by heat-inactivated A. pleuropneumoniae. Inactivated bacteria and bacteria in the presence of chloramphenicol each had no influence on the oxidative activity of neutrophils. In contrast, a heat-labile factor in A. pleuropneumoniae culture supernatants stimulated and inhibited the oxidative activity of the neutrophils in a dose-dependent manner. Undiluted and low dilutions of culture supernatants were toxic for the phagocytes, while high dilutions stimulated the oxygen radical production of the neutrophils. These effects were neutralized with homologous convalescent-phase pig sera. In order to investigate whether the heat-labile factors in the culture supernatant could be cytolysins, we repeated the experiments with cytolysin II and cytolysin III produced by recombinant Escherichia coli. It was demonstrated that stimulation and inhibition could be reproduced by both cytolysins. In conclusion, the observations made in this study showed that A. pleuropneumoniae secretes heat-labile metabolites that stimulate neutrophil-oxidative metabolism at relatively low concentrations and kill the neutrophils at higher concentrations. Cytolysins may be responsible, at least in part, for these effects.
A novel neutrophil-activating factor released by Trichomonas vaginalis
Authors: Shaio M F , Lin P R , Lee C S , Hou S C , Tang P , Yang K D
Published: January 2020

We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 10(7) T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37 degrees C for 30 min. With a multichamber chemotactic assay, we found that there were 112 +/- 15 migrated neutrophils (mean +/- standard deviation, n = 7) for STV and 11 +/- 4 for HBSS per high-power field (x 400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100 degrees C or deproteinization by treatment with proteinase K at 65 degrees C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (< 20 pg/ml) or prostaglandin E2 (< 8 pg/ml) was found in the STV by radioimmunoassay. Production of LTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 microM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells.
Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans
Authors: Palma C , Cassone A , Serbousek D , Pearson C A , Djeu J Y
Published: January 2020

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.
Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase
Authors: Burne R A , Penders J E
Published: January 2020

The complete nucleotide sequence (5,010 bp) of the fructanase gene (fruA) and flanking regions of the chromosome of Streptococcus mutans GS-5 was determined. The fruA gene appears to be the sole transcript arising from a proximal promoter. The presumed precursor of the secreted FruA protein consists of 1,423 amino acids, and it has an M(r) of 158,656 and a pI of 4.82. The N terminus of FruA has characteristics in common with signal peptides of gram-positive organisms. The C terminus consists of a serine- and threonine-rich region, followed by the peptide LPDTGD, 4 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the wall-spanning region, the LPXTGX consensus sequence, and the membrane-spanning domains of surface-associated proteins of gram-positive cocci. The FruA protein has significant homology with the Bacillus subtilis levanase (SacC), the Bacteroides fragilis levanase (ScrL), yeast invertases, and a number of other beta-fructosidases but not with fructosyltransferase, glucosyltransferases, or glucan-binding proteins of oral streptococci. Genes with homology to fruA were detected in S. mutans serotype c, e, and f strains, Streptococcus rattus, Streptococcus salivarius, and Streptococcus sanguis. A deletion derivative of FruA lacking the C-terminal 437 amino acids was still functional and could hydrolyze beta-(2,6)- and beta-(2,1)-linked sugars, but with altered preference for substrates. The data begin to define functional domains of the FruA protein and potential regulatory sites for induction, repression, growth rate control, and posttranslational localization of this multifunctional enzyme.
Polymorphism of the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi metacyclic trypomastigotes
Authors: Mortara R A , da Silva S , Araguth M F , Blanco S A , Yoshida N
Published: January 2020

In this study, we examined the immunochemical properties of the 35- and 50-kDa (35/50-kDa) surface glycoconjugates expressed on the surface of metacyclic trypomastigotes of Trypanosoma cruzi using three different monoclonal antibodies directed to this component. The 35/50-kDa surface antigen was expressed by metacyclic trypomastigotes of 11 different strains of T. cruzi and displayed a significant degree of molecular polymorphism. Results of immunoblotting and complement-mediated lysis were consistent with such diversity. Different monoclonal antibodies reacted with distinct epitopes on the 35/50-kDa antigen, which is resistant to proteases and behaves as an amphiphilic component.
Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare
Authors: Ogata K , Linzer B A , Zuberi R I , Ganz T , Lehrer R I , Catanzaro A
Published: January 2020

We have examined the activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare. M. avium-M. intracellulare at 2.5 x 10(6)/ml or 2.5 x 10(8)/ml was cultured in the presence of defensins at 37 degrees C from 4 to 48 h. After incubation, CFU were enumerated. Human neutrophil peptide 1 (HNP-1) at 5 micrograms/ml had the ability to kill M. avium-M. intracellulare. Treatment with HNP-1 resulted in significant (96.3 to 97.7%) killing of M. avium-M. intracellulare, even after taking clumping into consideration. This activity was not affected by the presence of calcium (0.5 and 1.0 mM), magnesium (0.5 and 1.0 mM), or sodium chloride (25, 50, and 100 mM). The optimal pH for bactericidal activity was higher than 5. We tested numerous M. avium-M. intracellulare strains, and HNP-1 was successful in killing every strain, although the degree of killing varied among them (34.2 to 87.2%). Additionally, this activity was independent of colonial morphology. We also examined the activity of HNP-2 and HNP-3 against M. avium-M. intracellulare and found that they were as effective in killing M. avium-M. intracellulare as HNP-1 was. These observations suggest that defensins may play an important role in the host defense against M. avium-M. intracellulare.
Characterization of a fucoside-binding adhesin of Candida albicans
Authors: Tosh F D , Douglas L J
Published: January 2020

Candida albicans GDH 2346 produces extracellular polymeric material (EP) which contains a mannoprotein adhesin with a lectin-like affinity for fucose-containing glycosides. EP isolated from culture supernatants of this strain was used as starting material for purification of the adhesin. The purification protocol involved a stepwise treatment of EP with N-glycanase, papain, and dilute alkali to cleave the protein and carbohydrate portions of the mannoprotein molecule. Fucoside-binding protein fragments were then recovered by affinity adsorption with the trisaccharide determinant of the H (type 2) blood group antigen which terminates in a residue of L-fucose. The purified adhesin was devoid of carbohydrate and inhibited yeast adhesion to buccal epithelial cells 221 times more efficiently, on a protein weight basis, than did EP. Adhesion inhibition reached a maximum of 78 to 80% at an adhesin concentration of 10 micrograms ml-1. Our results indicate that this protein is the major adhesin of yeast-phase cells of C. albicans GDH 2346 but that one or more secondary adhesion mechanisms may operate.
Dissemination of enteric Mycobacterium avium infections in mice rendered immunodeficient by thymectomy and CD4 depletion or by prior infection with murine AIDS retroviruses
Authors: Orme I M , Furney S K , Roberts A D
Published: January 2020

This study shows that infection of mice with the murine AIDS virus LP-BM5 or Du5H profoundly depressed the capacity of splenic T cells from these animals to respond to the T-cell mitogen phytohemagglutinin or concanavalin A or to alloantigens. Similar effects were also observed if mice were thymectomized and then infused with monoclonal anti-CD4 antibody (TxCD4- mice). When such mice were infected intravenously with Mycobacterium avium, growth of the infection was markedly exacerbated in the TxCD4- mice or in mice given murine AIDS virus 2 months earlier. In view of these data, we then investigated whether such treatments might cause dissemination of M. avium following enteric implantation of bacteria into the mouse cecum; this route was chosen in an attempt to model events in AIDS patients, in which the gut appears to be one of the major portals of M. avium infection. In this model, the entry and hematogenous dissemination of four clinical isolates of M. avium were monitored against time and found to be accelerated and enhanced in T-cell-deficient mice. In view of this finding, these novel approaches for enteric infection that use immunodeficient mice are presented as potential new models for the evaluation of immunotherapy and chemotherapy in a setting that bears some similarity to events believed to occur in AIDS patients.
Altered synthetic response of Campylobacter jejuni to cocultivation with human epithelial cells is associated with enhanced internalization
Authors: Konkel M E , Cieplak W
Published: January 2020

Campylobacter jejuni has been shown to bind to and enter epithelial cells in culture. The interaction of C. jejuni with INT 407 epithelial cells was examined to determine whether bacterial protein synthesis is required for either binding or internalization. Chloramphenicol, a selective inhibitor of bacterial protein synthesis, significantly reduced the internalization, but not binding, of C. jejuni compared with untreated controls as determined by protection from gentamicin. Electrophoretic analysis of metabolically labeled proteins revealed that C. jejuni cultured with INT 407 cells synthesized 14 proteins that were not detected in organisms cultured in medium alone. The inhibitory effect of chloramphenicol on internalization was reduced by preincubation of C. jejuni with INT 407 cells. The results indicate that C. jejuni, like some other enteric pathogens, engages in a directed response to cocultivation with epithelial cells by synthesizing one or more proteins that facilitate internalization and suggest that this phenomenon is relevant to the pathogenesis of enteritis caused by C. jejuni.
Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C
Authors: Crane J K , Wehner M S , Bolen E J , Sando J J , Linden J , Guerrant R L , Sears C L
Published: January 2020

The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family.
The arginine-dependent cytolytic mechanism plays a role in destruction of Naegleria fowleri amoebae by activated macrophages
Authors: Fischer-Stenger K , Marciano-Cabral F
Published: January 2020

Mouse peritoneal macrophages activated by different immunomodulators (Mycobacterium bovis bacillus Calmette-Guérin or Propionibacterium acnes) destroy Naegleria fowleri amoebae by a contact-dependent process and by soluble cytolytic molecules secreted by macrophages in response to lipopolysaccharide. The goal of this study was to determine whether the arginine-dependent cytolytic mechanism which results in the production of nitric oxide from arginine by activated macrophages destroys the amoebae. Amoebicidal activity of activated macrophages was determined by coculturing macrophages with N. fowleri amoebae radiolabeled with 3H-uridine. The percent specific release of radiolabel was used as an index of cytolysis of the amoebae. The inhibitors NG-monomethyl-L-arginine and arginase were used to determine whether the arginine pathway was a major effector mechanism responsible for amoebicidal activity of activated macrophages. Both the arginine analog NG-monomethyl-L-arginine and arginase, which breaks down arginine, decreased macrophage amoebicidal activity. Addition of arginine to arginine-free medium restores amoebicidal activity to activated macrophage cultures. These results demonstrate that the arginine pathway is an important mechanism for the destruction of susceptible N. fowleri amoebae.
Respiratory epithelial cell invasion by group B streptococci
Authors: Rubens C E , Smith S , Hulse M , Chi E Y , van Belle G
Published: January 2020

Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period; however, the pathogenesis of this infection is poorly understood. We investigated the ability of GBS to enter epithelial cells in culture. Two strains of GBS were capable of invading immortalized respiratory epithelial cell lines in vitro at different levels, suggesting strain differences in invasiveness. Intracellular replication was not observed. Invasion required actin microfilaments but not microtubular cytoskeletal elements. Active bacterial protein, DNA, and RNA syntheses were required for invasion. These findings are consistent with our previous observation of intracellular GBS in the lungs of infected primates. We hypothesize that this organism may access the bloodstream by direct invasion of the epithelial cell barrier.