bluuebunny/crossref_metadata_2025 huggingface dataset:
Susceptibility to reinfection after a primary chlamydial genital infection
Authors: Rank R G , Batteiger B E , Soderberg L S
Published: January 2020

Female guinea pigs which had been infected genitally with the agent of guinea pig inclusion conjunctivitis were challenged at various times after infection with fresh inocula to determine the duration of immunity resulting from the primary infection. At 30 days after infection, most guinea pigs were resistant to reinfection, as indicated by the inability to isolate chlamydiae from cervical swabs. However, at 77, 155, and 294 days, all animals became reinfected, although the course of the infection was abbreviated and of lower intensity. When various immune parameters were examined, a decrease in antibodies in both serum (immunoglobulin G [( IgG]) and genital secretions (IgA, IgG) was observed after 30 days. A decrease in antibodies to the major outer membrane protein and an 84K component was noted in serum. In genital secretions, IgA antibodies to all major chlamydial components declined markedly after 30 days. Cell-mediated immunity as measured by proliferation of peripheral blood lymphocytes to guinea pig inclusion conjunctivitis antigen also was at a peak response 30 days after infection and decreased thereafter. Thus, loss of complete immunity could not be associated with a particular immune parameter. When genital secretions were examined 14 days after the challenge infection, IgA antibody levels to the lipopolysaccharide and 61K protein components had increased in intensity, whereas other antibodies were relatively low. In addition, complete immunity to a third infection was not increased in duration when animals had recovered from two previous genital infections.
Effect of small cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane
Authors: Viljanen P , Koski P , Vaara M
Published: January 2020

Defensins are small cationic antibacterial peptides that are abundant in polymorphonuclear leukocytes from human and other sources (T. Ganz, M. Selsted, D. Szklarek, S. Harwig, K. Daher, D. F. Bainton, and R. J. Lehrer, J. Clin. Invest. 76:1427-1435, 1985). We studied whether subinhibitory concentrations of defensins increase the outer membrane (OM) permeability of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to hydrophobic probes, as do many other polycations that have been studied previously. Throughout the study, we used polymyxin B nonapeptide (PMBN) as a reference peptide. PMBN has a known potent OM permeability-increasing action. As a sharp contrast to PMBN, subinhibitory concentrations of defensins did not permeabilize (or, under some test conditions, permeabilized very slightly) the OM to the probes that were used (rifampin and Triton X-100). At bacteriostatic or bactericidal defensin concentrations, some degree of synergism with rifampin was seen.
Further characterization of the curative antibodies in Trypanosoma musculi infection
Authors: Wechsler D S , Kongshavn P A
Published: January 2020

The ability of immune plasma (IP) taken from different donor strains of mice to cure Trypanosoma musculi infection in various recipient mouse strains, when given during the plateau phase of infection, was examined. C57BL/6, B10.A/SgSn, B10.D2/oSn, B10.D2/nSn, DBA/2, and BALB/c strains could be cured of parasitemia (giving 0.4 to 0.8 ml of IP per mouse), whereas A/J and C3H/HeN strains could not (giving up to 1.2 ml of IP per mouse). Noncure appeared to be associated with the high-plateau parasitemias (approximately 10(8] that developed in the latter strains since IP administered early in infection, when the parasite burden was similar to the plateau parasitemias (approximately 10(6] of strains that could be cured, was at least partially effective in A/J and C3H/HeN mice. The IP of any strain tested (C57BL/6, B10.D2/oSn, B10.D2/nSn, DBA/2, A/J, or C3H/HeN) could bring about elimination of trypanosomes in strains able to be cured. The potency of IP from different strains varied, being greater in the strains that developed higher-plateau parasitemias. Potency of IP appears to correlate positively with the titers of trypanosome-specific antibody of the immunoglobulin G2a isotype (the curative antibody). The role of the late-acting complement components was examined. In C5-deficient mice the course of infection was normal, although the elimination phase was delayed by a few days. Cure of parasitemia by IP administered during the plateau phase was equally effective in the presence or absence of C5 in either the donor or the recipient. When tested in vitro, however, IP only exhibited antitrypanosomal activity when added to infected blood taken from C5-sufficient strains of mice. We conclude that in vitro, under the conditions used in the assay, antibody-mediated destruction of the trypanosomes is brought about by complement-mediated lysis. This process, although it probably occurs to some extent, is unlikely to be the major mechanism of trypanosome elimination in vivo.
Effect of intravenous silica on the course of Nocardia asteroides pneumonia
Authors: Pomeroy C , Filice G A
Published: January 2020

Silica, a known toxin of mononuclear phagocytes, was administered intravenously to mice during Nocardia asteroides pneumonia. Mice that received silica had a sevenfold decrease in the number of peripheral blood monocytes and developed more severe N. asteroides pneumonia than control mice. Lung histology in mice that received silica resembled that of mice with impaired cell-mediated immunity. These results are most consistent with the explanation that silica injures blood monocytes and impairs their contributions to pulmonary host defense.
Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages
Authors: Buchmeier N A , Heffron F
Published: January 2020

Salmonella typhimurium survives within macrophages and causes a fatal infection in susceptible strains of mice. A number of S. typhimurium mutants that contain Tn10 insertions in genes which are necessary for survival within the macrophage have been isolated. To demonstrate the importance of each gene in intracellular survival, the mutations were transduced into a smooth-strain background and the ability to survive intracellularly was assayed in five different populations of macrophages. The majority of the original macrophage-sensitive mutants retained the macrophage-sensitive phenotype in the smooth-strain background. The ability to survive or grow within macrophages varied with both the source of macrophages and the individual mutants. S. typhimurium grew best in the macrophage-like cell line J774, survived at moderate levels in splenic and bone marrow-derived macrophages, and was killed most efficiently in peritoneal macrophages. Macrophage-sensitive mutants transduced into a smooth background were also less virulent than the parent, with a 50% lethal dose of 2 to 5 logs greater than that of the parental strain. These experiments demonstrate that survival of S. typhimurium within macrophages varies with the source of cells, with a distinct ability to survive in macrophages from mouse spleens, where S. typhimurium grows rapidly. These experiments also demonstrate the heterogeneity in intracellular survival among the various macrophage-sensitive mutants, which may reflect the relative importance of the individual mutated genes in survival within macrophages.
The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease
Authors: Miller V L , Farmer J J , Hill W E , Falkow S
Published: January 2020

Yersinia enterocolitica is a heterogeneous group of organisms with more than 50 serotypes and several biotypes. Only a few of these serotypes cause gastrointestinal disease in otherwise healthy hosts; these serotypes are the pathogenic serotypes. Although Y. enterocolitica requires a high-molecular-weight plasmid to cause disease, chromosome-encoded determinants are required for the full expression of virulence. The ability of Yersinia spp. to invade eucaryotic cells is thought to be a virulence factor, because nonpathogenic serotypes are noninvasive in animals and in tissue culture cell models. Current evidence indicates that invasion ability is chromosome encoded. We recently reported cloning two loci, inv and ail, from Y. enterocolitica O8 strain 8081c that allow Escherichia coli to invade tissue culture cells. We investigated the link between invasion in an in vitro tissue culture invasion (TCI) model and hybridization to probes derived from the two invasion loci, inv and ail. We examined 177 Yersinia strains. Strains of serotypes and species associated with disease were TCI+, whereas strains of serotypes and species not associated with disease were TCI-. Only TCI+ strains had DNA homologous to probes derived from ail. All strains (TCI+ and TCI-) had DNA homologous to probes derived from inv, but there were certain restriction fragment-linked polymorphisms that were associated primarily with TCI+ strains. These observations held true for strains epidemiologically associated with disease. Both the inv and ail loci were found to be clearly located on the chromosome. No other genera, including other invasive organisms, had DNA homologous to inv or ail. These data support the hypothesis that the ail locus encodes a Y. enterocolitica invasion factor that may be involved in pathogenesis.
Comparative analysis of the genomes of intestinal spirochetes of human and animal origin
Authors: Coene M , Agliano A M , Paques A T , Cattani P , Dettori G , Sanna A , Cocito C
Published: January 2020

The aim of the present work was to compare the genomes of 21 strains of intestinal spirochetes, which were isolated from patients suffering intestinal disorders, with those of Treponema hyodysenteriae (strain P18), the known etiological agent of swine dysentery (bloody scours), and of a nonpathogenic strain (M1) of Treponema innocens. The percent guanine-plus-cytosine value of the 23 DNAs was found to be 25.5 to 30.1, as determined by a double-labeling procedure based on nick-translation by DNA polymerase I. The genome size of two spirochetal strains, of human and porcine origin, was found to be similar (4 x 10(6) base pairs) and close to that of the reference bacterium Escherichia coli (4.2 x 10(6) base pairs). Restriction analysis showed the presence of two modified bases in spirochetal DNA. Methyladenine was present in the GATC sequence of DNA from 15 spirochetes of human origin, and methylcytosine was present in several sequences occurring in all strains. The DNA of T. hyodysenteriae displayed a 30 to 100% homology with respect to that of 21 spirochetes from humans, thus suggesting the occurrence of a genetic heterogeneity in the latter group. These data indicate that the intestinal spirochetes analyzed in the present work are related; hence there is a possibility of domestic animals being reservoirs of microorganisms pathogenic for humans. A classification of intestinal treponemes into subgroups has been proposed on the basis of restriction analysis and hybridization experiments.
Effects of divalent cations and saccharides on Vibrio metschnikovii cytolysin-induced hemolysis of rabbit erythrocytes
Authors: Miyake M , Honda T , Miwatani T
Published: January 2020

Divalent cations and polysaccharides such as inulin and dextran reversibly inhibited hemolysis of rabbit erythrocytes caused by Vibrio metschnikovii cytolysin. On the basis of the 50% inhibitory doses, the cations were divided into two groups, group I (Cd2+, Cu2+, Ni2+, Sn2+, and Zn2+) and group II (Ba2+, Ca2+, Co2+, Mg2+, Mn2+, and Sr2+). Neither divalent cations nor polysaccharides interfered with the binding of toxins to the erythrocyte membrane. Group I cations disturbed tetramer formation of cytolysin on the cytolysin-lysed erythrocyte membrane, although group II cations and dextran did not affect the process. Erythrocytes treated with cytolysin in the presence of group II cations or dextran lysed after transfer to toxin- and inhibitor (group II cations or dextran)-free buffer at both 37 and 4 degrees C. However, erythrocytes treated in the presence of group I cations lysed at 37 degrees C but not at 4 degrees C, indicating that group I cations block the temperature-dependent lesion (tetramer)-forming step subsequent to the binding of cytolysin to the erythrocytes. The cytolysin-treated erythrocytes swelled in a colloid osmotic manner, and the swelling was preceded by the binding and the lesion-forming steps. It is also suggested that the lysis of the erythrocytes proceeds in a temperature-independent manner and that the cytolysin does not bind to the erythrocytes at 4 degrees C. These findings suggest that the sequence of V. metschnikovii cytolysin-induced hemolysis is defined by three steps: (i) a temperature-dependent binding step, (ii) a temperature-dependent lesion-forming step, and (iii) a temperature-independent lysis step.
Effect of oral immunization with Pseudomonas aeruginosa on the development of specific antibacterial immunity in the lungs
Authors: Freihorst J , Merrick J M , Ogra P L
Published: January 2020

Oral administration of Pseudomonas aeruginosa to rats elicited a systemic and mucosal antibody response in the intestinal and respiratory tracts. The antibody response did not influence the course of the disease when immunized animals were subsequently challenged by the introduction of viable bacteria into the lungs.
Immunochemical and structural characterization of a serotype-specific polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b)
Authors: Amano K , Nishihara T , Shibuya N , Noguchi T , Koga T
Published: January 2020

A serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) was extracted from whole cells by autoclaving. The extract was purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. The purified polysaccharide antigen formed a single precipitin line with anti-type b serum but not with anti-type a serum and anti-type c serum. The antigen was composed of 43.9% L-rhamnose, 49.1% D-fucose, and a trace amount of fatty acid. Methylation analysis, Smith degradation, and optical rotation data showed that the antigen was a polymer consisting of a disaccharide repeating unit, ----3)-alpha-D-fucopyranosyl-(1----2)-beta-L-rhamnopyranosyl-(1----. In quantitative precipitin inhibition tests, D-fucose and L-rhamnose showed very low inhibition, but the partial hydrolysate of the purified antigen was an effective inhibitor, suggesting that the serotype b specific antiserum recognizes the larger oligosaccharide units.
Adhesion to and invasion of HEp-2 cells by Campylobacter spp
Authors: Konkel M E , Joens L A
Published: January 2020

Twenty-one isolates were tested for their ability to adhere to and invade HEp-2 cells in vitro. Of the 21 organisms tested, 2 did not invade the HEp-2 cells, and 1 of these did not adhere to the epithelial cells. Campylobacter jejuni clinical isolates were more invasive than the nonclinical strains that were tested. When HEp-2 cells were treated with cytochalasin B, the invasiveness of C. jejuni was reduced, indicating active participation of the host cell in the uptake of these organisms. The number of intracellular C. jejuni isolates decreased when Campylobacter whole-cell lysates were absorbed onto HEp-2 cell monolayers. Experiments were also conducted to identify the functional sites of the antigens responsible for expression of Campylobacter invasion. Oxidation of lysates with sodium meta-periodate significantly affected its inhibitory capacity. This implies that the Campylobacter invasive ligand appears to be dependent upon an intact carbohydrate moiety.
Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells
Authors: Arroyo R , Alderete J F
Published: January 2020

The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T. vaginalis cytadherence. Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone. Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment. Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T. vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T. vaginalis in the presence and absence of TLCK were identical. Kinetics of TLCK-mediated inhibition of cytadherence of other T. vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286. Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence. Finally, treatment of T. vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity. These data show for the first time the involvement of T. vaginalis cysteine proteinases in parasite attachment to human epithelial cells. These results have implications for future pharmacologic intervention at a key step in infection.
Identification of a chromosomal locus for expression of lipopolysaccharide epitopes in Haemophilus influenzae
Authors: Weiser J N , Lindberg A A , Manning E J , Hansen E J , Moxon E R
Published: January 2020

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is able to display an extensive repertoire of different LPS structures through the loss and acquisition of multiple oligosaccharide epitopes in various combinations. This marked heterogeneity of LPS molecules has complicated the analysis of the structure of LPS and its role in pathogenesis. A genomic library was screened for the ability to transform H. influenzae to express novel LPS epitopes defined by reactivity with oligosaccharide specific monoclonal antibodies. A chromosomal locus, lic-1, involved in expression of at least three different epitopes (recognized by monoclonal antibodies 4C4, 12D9, and 6A2), was identified on a 5.6-kilobase restriction endonuclease fragment. Transformation of H. influenzae with subclones from within lic-1 was used to generate a series of isogenic and phenotypic variants. All transformants displayed phase variation for their newly acquired epitopes. Altered binding specificities of LPS with monoclonal antibodies correlated with changes in sugar compositional analysis. The expression of two epitopes was eliminated by introduction of site-specific mutations in lic-1, confirming the role of lic-1 in oligosaccharide biosynthesis.
Differences in adhesion of Pseudomonas aeruginosa to mucin glycopeptides from sputa of patients with cystic fibrosis and chronic bronchitis
Authors: Ramphal R , Houdret N , Koo L , Lamblin G , Roussel P
Published: January 2020

Pseudomonas aeruginosa is the most prominent colonizer of the respiratory tract of patients with cystic fibrosis, but it is not known why this occurs. P. aeruginosa adheres to mucins from normal individuals, but mucins from cystic fibrosis patients have not been studied. To compare adhesion to mucins from cystic fibrosis with other mucins, we prepared highly glycosylated mucin glycopeptides from cystic fibrosis and chronic bronchitis patients by ion-exchange and gel-filtration chromatography and measured the adhesion of P. aeruginosa 1244 to these glycopeptides. We found (i) that the most mucinlike glycopeptides from P. aeruginosa-infected cystic fibrosis sputa showed less bacterial adhesion than did the corresponding bronchitis samples, (ii) that the most adhesive activity in cystic fibrosis samples came from a fraction that contains O and N glycopeptides and may be in part a degradation product of P. aeruginosa infection, and (iii) that highly glycosylated glycopeptides of the most acidic species (sialylated and sulfated) showed no adhesion at all. A single cystic fibrosis sample not infected by P. aeruginosa showed better binding in the adhesion-positive fractions than did the infected sputa. These studies suggest that cystic fibrosis mucins may be altered after infection is established, resulting in less binding to some fragments. However, since the clinical picture shows heavy mucus colonization, other receptors, such as cellular glycolipids which have been shed into mucus, may be contributing to this colonization.
Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis
Authors: Borremans M , de Wit L , Volckaert G , Ooms J , de Bruyn J , Huygen K , van Vooren J P , Stelandre M , Verhofstadt R , Content J
Published: January 2020

We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140- or 125-kDa beta-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, M. Weckx, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promoter sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG alpha-antigen (K. Matsuo, R. Yamaguchi, A. Yamazaki, H. Tasaka, and T. Yamada, J. Bacteriol. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B- and T-cell epitope regions on the 32-kDa antigen.
Plasmodium falciparum schizont sonic extracts suppress lymphoproliferative responses to mitogens and antigens in malaria-immune adults
Authors: Riley E M , Jobe O , Blackman M , Whittle H C , Greenwood B M
Published: January 2020

Cellular immune responses to malaria antigens are suppressed during acute Plasmodium falciparum infection, and evidence from both murine and human studies suggests that parasite-derived factors may be directly immunosuppressive. In this study we have shown that P. falciparum schizont sonic extract will suppress in vitro lymphoproliferative responses to purified malaria antigens and other soluble antigens. The degree of suppression appears to correlate with the level of the lymphoproliferative response to the schizont preparation and is correspondingly more marked in malaria-immune donors than in nonimmune individuals. The effect can be transferred with primed mononuclear cells and is partially abrogated by removal of CD8+ lymphocytes. The suppressive component of the schizont preparation is nondialyzable and partially heat labile and comigrates with hemoglobin-derived proteins in the molecular mass range 10 to 20 kilodaltons.
Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis
Authors: Ridderhof J C , Barnes R C
Published: January 2020

We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle.
Inhibition of coaggregation between Fusobacterium nucleatum and Porphyromonas (Bacteroides) gingivalis by lactose and related sugars
Authors: Kolenbrander P E , Andersen R N
Published: January 2020

The coaggregation of Fusobacterium nucleatum PK1594 and Porphyromonas (Bacteroides) gingivalis PK1924 was inhibited equally well by lactose, N-acetyl-D-galactosamine, and D-galactose, which caused 50% inhibition of coaggregation at 2 mM sugar concentration. Other sugars such as D-galactosamine, D-fucose (6-deoxy-D-galactose), and alpha-methyl- and beta-methyl-D-galactosides also inhibited coaggregation. Sugar specificity was apparent, since neither L-fucose, L-rhamnose, N-acetyl-D-glucosamine, nor N-acetylneuraminic acid was an inhibitor. Protease treatment of the fusobacterium completely abolished coaggregation, whereas it had no effect on the coaggregating activity of the porphyromonad. Although numerous lactose-inhibitable coaggregating pairs are known to occur among gram-positive bacteria, this report and the accompanying survey (P. E. Kolenbrander, R. N. Andersen, and L. V. H. Moore, Infect. Immun. 57:3194-3203, 1989) are the first studies demonstrating the extensive nature of this type of interaction between gram-negative human oral bacteria. The significance of galactoside-inhibitable coaggregations between these two potential periodontal pathogens is discussed.
Identification of an immunodominant antigenically conserved 32-kilodalton protein from Cowdria ruminantium
Authors: Jongejan F , Thielemans M J
Published: January 2020

Western blotting (immunoblotting) of Cowdria ruminantium antigens with goat or mouse antiserum identified a periodate-resistant, proteinase K-sensitive immunodominant antigen of 32,000 daltons. This protein, designated Cr32, could be demonstrated in goat choroid plexus infected with one of two different Cowdria stocks. Antisera against nine different Cowdria stocks from Africa and the Caribbean region recognized Cr32, which indicates that this protein contains conserved antigenic determinants.
Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II)
Authors: Lee S F , Progulske-Fox A , Erdos G W , Piacentini D A , Ayakawa G Y , Crowley P J , Bleiweis A S
Published: January 2020

The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.
Binding of pertussis toxin to eucaryotic cells and glycoproteins
Authors: Witvliet M H , Burns D L , Brennan M J , Poolman J T , Manclark C R
Published: January 2020

The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc----Gal beta 4GlcNAc) sequence on surface macromolecules. In addition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined corresponding binding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin to cellular components and purified glycoproteins was observed. The two dimers bound to a number of glycoproteins containing N-linked oligosaccharides but not O-linked oligosaccharides, and differences in the binding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin molecule contains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins.
Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV
Authors: Knutton S , McConnell M M , Rowe B , McNeish A S
Published: January 2020

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.
Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum
Authors: Isaacs R D , Hanke J H , Guzman-Verduzco L M , Newport G , Agabian N , Norgard M V , Lukehart S A , Radolf J D
Published: January 2020

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.
Characterization of coaggregation between Bacteroides gingivalis T22 and Fusobacterium nucleatum T18
Authors: Kinder S A , Holt S C
Published: January 2020

Bacterial adherence is a key factor in the colonization of the oral ecosystem, yet little is known about the mechanisms by which the pathogen Bacteroides gingivalis adheres in the periodontal environment. We examined the ability of strains of B. gingivalis to coaggregate with selected microorganisms isolated from the subgingival microbiota of the cynomolgus monkey. A strong interaction was demonstrated between strains of B. gingivalis and Fusobacterium nucleatum, whereas less pronounced or no interaction was observed with other oral isolates. Electron microscopic examination of coaggregates revealed large masses of bacteria, in which the fusiform F. nucleatum T18 and coccobacillary B. gingivalis T22 cells formed a woven pattern. To investigate this interaction and the nature of the bacterial cell surface molecules involved, we used a microcoaggregation assay. Galactose and galactose-related sugars blocked coaggregation, in contrast with the lack of effect of glucose or glucose-related sugars. The ability of F. nucleatum T18 cells to coaggregate was diminished by pretreatment with pronase. Pretreatment of B. gingivalis T22 cells with pronase resulted in an inhibition of coaggregation, whereas pretreatment with sodium metaperiodate completely abolished coaggregation. These data suggest that the coaggregation between B. gingivalis T22 and F. nucleatum T18 represents a carbohydrate-lectin interaction, mediated by a galactose-containing carbohydrate on B. gingivalis T22 and a protein on F. nucleatum T18.
Otitis media in children: local immune response to nontypeable Haemophilus influenzae
Authors: Faden H , Brodsky L , Bernstein J , Stanievich J , Krystofik D , Shuff C , Hong J J , Ogra P L
Published: January 2020

Twenty children experienced 30 episodes of otitis media with effusion due to nontypeable (NT) Haemophilus influenzae in the first 2 years of life. The local and systemic immune responses to homologous strains of NT H. influenzae were determined by an immunodot assay. Strain-specific immunoglobulin G (IgG) antibody predominated in the middle ear fluid (MEF). It was detected in 91% of the children, compared with IgM in 48% (P less than 0.005), IgA in 52% (P less than 0.005), and secretory IgA in 18% (P less than 0.005). The titer (log2) of NT H. influenzae-specific IgG antibody (mean +/- standard error, 8.2 +/- 0.1) exceeded the titers of IgM (3.4 +/- 0.1), IgA (3.7 +/- 0.1), and secretory IgA (1.2 +/- 0.3). NT H. influenzae-specific antibody was detected exclusively in MEFs of individuals who possessed homologous serum antibody. Although antibody titers in MEF declined over time, serum antibody titers remained stable. These data suggest that immunity to NT H. influenzae in the middle ear, in part, reflects systemic immunity. Whereas local antibody disappears after resolution of the infection, systemic antibody persists.
Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells
Authors: Kushnaryov V M , Sedmak J J
Published: January 2020

Electron microscopical immunocytochemistry and light microscopy were used to study the effect of Clostridium difficile enterotoxin A (EA) on cultured Chinese hamster ovary (CHO) cells. At 4 degrees C, immunocytochemically detected EA was randomly distributed along the plasma membrane; when cells were subsequently transferred to 37 degrees C, the EA moved into coated pits and coated vesicles within 2 min. Within 2 h of incubation at 37 degrees C with EA, the perinuclear cytoplasm of the CHO cells became highly refractile, and in 4 h, most of the cells were round; however, the majority of rounded cells excluded erythrosin B while remaining firmly attached to the culture dish plastic. When EA was removed from the cultured cells within 2 h, the cells returned to a normal morphology and formed a confluent monolayer. The nuclei of rounded cells were irregularly shaped; cytoplasmic intermediate filaments were collapsed toward the nucleus. Long bundles of parallel, 11-nm-diameter filaments appeared in the nuclei after 3 h of incubation with EA and disappeared by the fourth hour of incubation. Intoxicated cells did not undergo mitosis. Thus, EA was internalized, at least in part, by receptor-mediated endocytosis and subsequently affected the nuclei of CHO cells.
Purification and characterization of two Listeria ivanovii cytolysins, a sphingomyelinase C and a thiol-activated toxin (ivanolysin O)
Authors: Vazquez-Boland J A , Dominguez L , Rodriguez-Ferri E F , Suarez G
Published: January 2020

The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described.
Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli
Authors: Arthur M , Johnson C E , Rubin R H , Arbeit R D , Campanelli C , Kim C , Steinbach S , Agarwal M , Wilkinson R , Goldstein R
Published: January 2020

The pap, prs, pil, and hly operons of the pyelonephritic Escherichia coli isolate J96 code for the expression of P, F, and type 1 adhesins and the production of hemolysin, respectively; the afaI operon of the pyelonephritic E. coli KS52 encodes an X adhesin. Using different segments of these operons as probes, colony hybridizations were performed on 97 E. coli urinary tract and 40 fecal clinical isolates to determine (i) the presence in the infecting bacteria of nucleotide sequences related to virulence operons, and (ii) the phenotypic properties associated with such sequences. Coexpression of P and F adhesins encoded by pap-related sequences was detected more frequently among isolates from patients with pyelonephritis (32 of 49, 65%) than among those with cystitis (11 of 48, 23%; P less than 0.0001) or from fecal specimens (6 of 40, 15%; P less than 0.0001). Therefore, the expression of both adhesins appears to be critical in the colonization of the upper urinary tract. In contrast, afaI-related sequences were detected significantly more frequently among isolates from patients with cystitis, suggesting that this class of X adhesin may have a role in lower urinary tract infections. Urinary tract isolates differed from fecal isolates by a low incidence of type 1 adhesin expression among pil probe-positive isolates. hly-related sequences were only detected in pap probe-positive isolates. The frequency of hemolysin production among pap probe-positive isolates was not associated with a particular pattern of infection. The distribution of these virulence factors was similar in the presence or absence of reflux, indicating that structural abnormalities of the urinary tract did not facilitate colonization by adhesin-negative isolates.
Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein
Authors: Pál T , Newland J W , Tall B D , Formal S B , Hale T L
Published: January 2020

Escherichia coli K-12 hybrids carrying both the 220-kilobase plasmid and the purE-linked kcpA locus from Shigella flexneri expressed a 140-kilodalton (kDa) protein which was recognized by convalescent sera from monkeys infected with S. flexneri. These hybrids were tested for the ability to produce plaques in HeLa cell monolayers. Hybrid strains which carried both the 220-kilobase plasmid and the kcpA locus had a plaque-forming efficiency of at least 10(-4) PFU/CFU, whereas the plaque-forming efficiency of hybrids that carried only the shigella invasion plasmid ranged from undetectable to 10(-6). Variants were purified from the rare plaques formed by E. coli hybrids that carried only the shigella invasion plasmid. These plaque-purified strains also expressed the 140-kDa protein, and they had a plaque-forming efficiency of at least 10(-4). Transduction of the purE locus from a plaque-purified hybrid into a non-plaque-forming E. coli K-12 strain did not alter the phenotype of the recipient, but conjugation of the shigella invasion plasmid into this transductant reconstituted both expression of the 140-kDa protein and the plaque-forming phenotype. Invasive E. coli K-12 hybrids carrying only the shigella invasion plasmid remained localized within discrete areas of the HeLa cell cytoplasm, whereas hybrids that also carried the S. flexneri kcpA locus grew in a dispersed pattern throughout the host cell cytoplasm. The dispersal of these organisms was inhibited by cytochalasin D, which suggested that host cell microfilaments may play a role in the intracellular spread of enteroinvasive pathogens.
Separate and combined effects of recombinant interleukin-1 alpha and gamma interferon on antibacterial resistance
Authors: Kurtz R S , Young K M , Czuprynski C J
Published: January 2020

Our laboratory has previously reported that administration of murine recombinant interleukin 1 alpha (rIL-1 alpha) substantially enhanced the resistance of mice to Listeria monocytogenes infection. Other investigators have reported that gamma interferon (IFN-gamma) plays a pivotal role in antilisteria resistance. In the present study, we have defined doses of human rIL-1 alpha that enhanced the antilisteria resistance of mice. We then addressed the possibility that combined immunotherapy with rIL-1 alpha and recombinant IFN-gamma (rIFN-gamma) might result in an additive or synergistic enhancement of antibacterial resistance. Simultaneous administration of rIL-1 alpha and rIFN-gamma enhanced antilisteria resistance (at 3 days after infection) to a greater extent than did either cytokine alone, although the results did not imply a synergistic action between the two cytokines. Experiments which examined the effects of the timing of cytokine administration indicated that maximal protection was observed when rIL-1 alpha and rIFN-gamma were administered together concomitantly with the L. monocytogenes challenge. When we compared the separate and combined protective effects of rIL-1 alpha and rIFN-gamma throughout the course of a primary L. monocytogenes infection, we observed an additive effect of the two cytokines only at 3 days after challenge, the time at which the peak bacterial burden occurs in the spleens and livers of infected mice. Histopathological comparisons of livers and spleens from cytokine-treated and control listeria-infected mice verified that cytokine treatment reduced the severity of tissue damage in cytokine-treated listeria-infected mice. In an attempt to provide a potential mechanism for the protective effects of rIL-1 alpha and rIFN-gamma administration, we compared levels of colony-stimulating activity in sera from cytokine-treated and control listeria-infected mice. The highest levels of colony-stimulating activity were detected in sera from control listeria-infected mice; somewhat lower levels were found in sera from listeria-infected mice that received rIL-1 alpha and rIFN-gamma either alone or in combination.