bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the F41 fimbrial antigen of enterotoxigenic Escherichia coli by using monoclonal antibodies
Authors: van Zijderveld F G , Westenbrink F , Anakotta J , Brouwers R A , van Zijderveld A M
Published: January 2020

We produced monoclonal antibodies (MAbs) from 23 different murine hybridoma cell lines against the F41 fimbrial antigen of bovine and porcine enterotoxigenic Escherichia coli. Cell lines were created by fusing myeloma cells and spleen cells of mice that were immunized with either purified F41 or with Formalin-killed whole cells. The specificity of the MAbs to the F41 antigen was proven by enzyme-linked immunosorbent assays (ELISAs) and radioimmunoprecipitation tests. Epitope analysis with a competition ELISA revealed that the 23 MAbs recognized at least five epitopes. These results were corroborated by those of immunodiffusion tests, in which all possible combinations of two MAbs were tested against ultrasonically disintegrated F41 antigen. In a double-antibody sandwich ELISA, all peroxidase-conjugated MAbs bound to the F41 antigen of all 182 bacterial strains that were tested. Apparently, the epitopes recognized by the MAbs are highly conserved. Immunoelectron microscopy revealed that the MAbs were directed to fimbrial structures 3 to 4 nm in diameter and that the epitopes were equally distributed along the fimbriae. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed. The results of the radioimmunoprecipitation assay suggested that F41 fimbriae are composed of a single repeating 29,000-dalton protein subunit; however, we could not exclude the possibility of the existence of minor fimbrial components.
Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum
Authors: Radolf J D , Moomaw C , Slaughter C A , Norgard M V
Published: January 2020

Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.
Whole, submandibular, and parotid saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis
Authors: Komiyama K , Habbick B F , Tumber S K
Published: January 2020

The aggregation of mucoid and nonmucoid Pseudomonas aeruginosa by submandibular, parotid, and whole saliva from patients with cystic fibrosis (CF) and non-CF subjects was investigated. There were significant differences (P less than 0.01) in aggregation of mucoid and nonmucoid variants of P. aeruginosa by submandibular and whole saliva from CF patients and non-CF subjects. However, the differences in the parotid secretion were not as pronounced. Patients with CF who were colonized with P. aeruginosa demonstrated a significantly higher (P less than 0.05) percent aggregation of the mucoid variants by the submandibular secretion and of both mucoid and nonmucoid variants by whole saliva, compared with corresponding secretions from patients with CF not colonized with this pathogen. The parotid saliva aggregation activity was not markedly different for the two groups with CF. From patients with CF, whole saliva demonstrated a higher percent P. aeruginosa aggregation than did the submandibular saliva. In non-CF subjects, however, the percent aggregation of P. aeruginosa by submandibular saliva was higher than that by whole saliva. Our results indicate that the sero-mucous products of the submandibular gland have a more significant role in P. aeruginosa aggregation than the serous secreting parotid cells and that the submandibular secretion is possibly responsible for the differences in oral colonization by this pathogen in subjects with and without CF.
Actinomyces viscosus fibril antigens detected by immunogold electron microscopy
Authors: Ellen R P , Buivids I A , Simardone J R
Published: January 2020

Strains representing taxonomic clusters of Actinomyces viscosus and Actinomyces naeslundii were studied by indirect immunogold electron microscopy with either monospecific anti-type 1 and anti-type 2 rabbit antibodies or species-specific monoclonal antibodies. The monoclonal and anti-type 2 antibodies localized on long fibrils, whereas the anti-type 1 antibodies mostly localized close to the cell body or on shorter appendages.
Tumor necrosis factor alpha is a cytotoxin induced by murine Chlamydia trachomatis infection
Authors: Williams D M , Bonewald L F , Roodman G D , Byrne G I , Magee D M , Schachter J
Published: January 2020

A mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent) was used to demonstrate that whole spleen cells from both nude athymic mice (nu/nu) and heterozygous mice (nu/+) produced tumor necrosis factor alpha in vitro in response to mouse pneumonitis agent antigen. The tumor necrosis factor alpha measured in these supernatants by immunoassay was shown to have bioactivity in a cytotoxic assay in which uninfected target cells were used. This cytotoxicity was distinct from the gamma interferon-related cytotoxicity against C. trachomatis-infected targets that we described previously.
Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice
Authors: Groves M G , Kelly D J
Published: January 2020

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.
Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence
Authors: Grosskinsky C M , Jacobs W R , Clark-Curtiss J E , Bloom B R
Published: January 2020

Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.
Differential modulation of lymphocyte proliferative responses and lymphokine secretion in mice during development of immunity to Chlamydia psittaci
Authors: Guagliardi L E , Byrne G I , Paulnock D M
Published: January 2020

A murine model was utilized to study immune responses occurring during the period of acquisition of immunity to chlamydial infection. C3H (H-2k) mice were immunized by intramuscular injection of 5 x 10(3) viable Chlamydia psittaci elementary bodies (EBs) by a protocol which permits animals to survive an otherwise lethal intraperitoneal challenge 10 days later with the homologous chlamydial strain. Spleen cells assayed during the 10-day period of development to immunity showed depressed proliferative responses in vitro to the T-cell mitogen, concanavalin A, and also exhibited suppressor cell activity. Spleen cell mitogen responses returned to normal levels by 30 days postimmunization, concomitant with the detectable development in vitro of responses to chlamydia-specific antigen. In marked contrast to the reduced proliferative responses, mitogen-stimulated production of the T-cell-derived lymphokines interleukin-2 and gamma interferon by spleen cells from immunized animals was within the normal range at 10 days postimmunization, and supernatant fluids containing these products had both microbicidal and microbistatic effects on chlamydial organisms in vitro. These results demonstrate that independent regulation of T-cell proliferation and lymphokine production occurs in vivo as part of the development of an antigen-specific protective immune response. These results also suggest that such differential modulation of T-cell responses may contribute to the development of protective immunity to chlamydiae in mice, perhaps through limited T-cell clonal expansion coupled with early or preferential maturation of cytokine-secreting helper T cells.
Inactivation of human gamma interferon by Pseudomonas aeruginosa proteases: elastase augments the effects of alkaline protease despite the presence of alpha 2-macroglobulin
Authors: Horvat R T , Clabaugh M , Duval-Jobe C , Parmely M J
Published: January 2020

Pseudomonas aeruginosa alkaline protease (AP) has recently been shown to produce limited proteolysis of human gamma interferon (IFN-gamma) and thereby destroy the antiviral and macrophage-activating activities of the lymphokine. In the present study we describe some of the characteristics of Pseudomonas elastase (E) with regard to inactivation of human IFN-gamma. The inhibitory effect of E on IFN-gamma bioactivity differed from that of AP in that the direct effects of E were reduced in the presence of human serum. That this property of human serum was in large part attributable to the protease inhibitor alpha 2-macroglobulin (alpha 2-M) was suggested by the following observations: (i) methylamine treatment of serum reduced its effect on E, (ii) E interacted directly with alpha 2-M to induce a characteristic conformational change in the protease inhibitor, and (iii) preformed E-alpha 2-M complexes lacked IFN-gamma-degrading activity. Despite these findings, anti-E antiserum partially neutralized the effect that a Pseudomonas filtrate showed on IFN-gamma, suggesting that E contributes to the activity of bacterial filtrates. Treatment of IFN-gamma with E in the presence of a suboptimal concentration of AP resulted in an E dose-dependent inactivation of the lymphokine. Preformed E-alpha 2-M complexes, although ineffective by themselves at cleaving IFN-gamma, degraded the lymphokine, providing AP was also present in the reaction mixture. These data demonstrate that the destruction of small, biologically significant peptides by Pseudomonas proteases can involve protease-protease synergy that acts even in the presence of the serum protease inhibitor alpha 2-M.
Secretory immunoglobulin A response to Shiga toxin in rabbits: kinetics of the initial mucosal immune response and inhibition of toxicity in vitro and in vivo
Authors: Keren D F , Brown J E , McDonald R A , Wassef J S
Published: January 2020

Although the role of Shiga toxin in dysentery is unknown, the toxin is cytotoxic to HeLa cells, causes fluid secretion in rabbit intestine, and is lethal to rabbits and mice when injected parenterally. In the present study, rabbits received three weekly doses of Shiga toxin directly into chronically isolated ileal loops. Within a week, secretions from these loops contained immunoglobulin A (IgA) anti-Shiga toxin. The titer of IgA anti-Shiga toxin increased after weekly doses 2 and 3. Little IgG anti-Shiga toxin was present in loop secretions, although high titers of IgG anti-Shiga toxin were found in the sera. These loop secretions were able to neutralize the cytotoxic effects of Shiga toxin in the HeLa cell assay. The capacity to neutralize the cytotoxicity of the toxin correlated strongly with the IgA anti-Shiga toxin titer in these same secretions. Pooled immune loop secretions were also able to significantly reduce fluid accumulation in acutely ligated loops in rabbits, while loop secretions from control rabbits could not. Shiga toxin elicited a strong secretory IgA response upon application to the intestine. Further, the mucosal antibodies produced functioned to prevent the toxic effects of Shiga toxin both in vitro and in vivo.
Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components
Authors: Riesenfeld-Orn I , Wolpe S , Garcia-Bustos J F , Hoffmann M K , Tuomanen E
Published: January 2020

While there is considerable evidence that both interleukin-1 (IL-1) and tumor necrosis factor (TNF) are central mediators of inflammation caused by gram-negative bacteria and endotoxin, the roles of these two mediators in gram-positive infection are unknown. Pneumococcal infections are characterized by an intense inflammatory reaction in infected tissues. Current evidence suggests that the component of the pneumococcus which causes this inflammation in many body sites is the cell wall. We determined the ability of native pneumococcal cell wall, lipoteichoic acid, and cell wall subcomponents to stimulate secretion of IL-1 and TNF from human monocytes. Each pneumococcal cell surface component was found to have a different specific activity for induction of IL-1. Teichoication was an important determinant of this activity: teichoicated species were at least 10,000-fold more potent than endotoxin and 100-fold more potent than teichoic acid-free peptidoglycan. IL-1-inducing activity was greatly reduced by chemical alteration of the teichoic acid. In contrast to endotoxin, cell wall did not induce production of TNF. This dissociation of the production of IL-1 and TNF during the response of the human monocyte to pneumococcal surface components suggests that, in at least some circumstances, the mechanisms for generation of an inflammatory response to infection may be fundamentally different between gram-positive and gram-negative disease.
Effect of chemical modification of histidine and tyrosine residues in toxic shock syndrome toxin 1 on the serologic and mitogenic activities of the toxin
Authors: Kokan-Moore N P , Bergdoll M S
Published: January 2020

Modification of three or four of the five histidine residues in the toxic shock syndrome toxin 1 (TSST-1) with diethylpyrocarbonate did not inhibit the precipitin reaction of the modified TSST-1 with polyvalent antisera to the toxin. Monoclonal antibody 7T did not react with the modified TSST-1, but monoclonal antibody 8T did react with the toxin. Up to 50% of the mitogenic reaction of TSST-1 was inhibited by the histidine modification. Modification of one or two of the nine tyrosine residues in TSST-1 did not inhibit the precipitin reaction with polyclonal antisera to the toxin but did inhibit 85% of the mitogenic reaction.
Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis
Authors: Slagle D C , Cox R A , Kuruganti U
Published: January 2020

The cytokine tumor necrosis factor alpha (TNF-alpha) functions as an immunomodulatory protein and as a mediator of cachexia. We report that viable or Formalin-killed spherules of Coccidioides immitis induced the secretion of TNF-alpha by peritoneal-exudate cells from BALB/c mice. The identification of the cytokine as TNF-alpha was based on its lytic activity against the TNF-alpha-sensitive LS murine fibrosarcoma cell line but not the TNF-alpha-resistant LR cell line, its neutralization by rabbit anti-TNF-alpha, and its secretion by peritoneal cells having characteristics of macrophages. The induction of TNF-alpha was to spherules and not to contaminating lipopolysaccharide (endotoxin), as evidenced by the finding that polymyxin B, a reagent that blocks the TNF-alpha-inducing component of lipopolysaccharide, did not negate the production of TNF-alpha in response to spherules, whereas pretreatment of spherules with hyperimmune goat antiserum to spherulin neutralized the induction of TNF-alpha by these cells. The demonstration that C. immitis activates macrophages to secrete TNF-alpha in vitro is a new finding and warrants studies to determine whether this cytokine is produced during active coccidioidomycosis.
Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model
Authors: Miliotis M D , Koornhof H J , Phillips J I
Published: January 2020

The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture. Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria. As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time. Monolayers infected for 3 h were treated with antibiotics and either [14C]glutamine or [3H]leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured. A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid. This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time. None of the South African enteropathogenic E. coli isolates used in these studies produced Vero cytotoxin. These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E. coli.
Trypanosoma cruzi expresses diverse repetitive protein antigens
Authors: Hoft D F , Kim K S , Otsu K , Moser D R , Yost W J , Blumin J H , Donelson J E , Kirchhoff L V
Published: January 2020

We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship.
Transformation of fluoride resistance genes in Streptococcus mutans
Authors: Chansley P E , Kral T A
Published: January 2020

To study the nature of fluoride resistance in Streptococcus mutans, we transformed DNA extracted from fluoride-resistant mutants of S. mutans GS-5 into fluoride-sensitive cells of the same strain. Transformation with DNA from first-step mutants produced transformants with resistance to either 600 or 1,000 micrograms of sodium fluoride per ml, both of which are within the first-step resistance range (400 to 1,000 micrograms/ml). In five of six of these transformation experiments, however, the transformant resistance levels were greater than those of their respective DNA donors. Transformation with DNA from a second-step mutant resistant to 1,600 micrograms/ml resulted in transformants resistant to 600 micrograms/ml, similar to some transformants receiving DNA from first-step mutants. When a second-step mutant resistant to 3,000 micrograms/ml was used as a DNA donor, four different levels of resistance were seen in the transformants (600, 1,000, 1,500, and 2,000 micrograms/ml). In many cases, the growth rates of the transformants (first and second step) were faster than those of the DNA donors. Additionally, many of the transformants demonstrated abrupt shifts in growth rates at relatively low culture densities.
Determinants for thermoinducible cell binding and plasmid-encoded cellular penetration detected in the absence of the Yersinia pseudotuberculosis invasin protein
Authors: Isberg R R
Published: January 2020

Yersinia pseudotuberculosis inv mutants were analyzed for their ability to bind and penetrate mammalian cell lines. Strains defective for the production of invasin and cured of the Yersinia virulence plasmid pIB1 were extremely defective for entry into the HEp-2 cell line. inv strains harboring the virulence plasmid partially overcame this defect, indicating that the virulence plasmid mediates an invasin-independent pathway for low-level entry into cultured cells. Plasmid-cured inv mutants were able to attach efficiently to mammalian cells after bacterial culture at 37 degrees C but not after culture at a lower temperature. The enhanced cellular binding of inv mutants grown at 37 degrees C did not result in efficient cellular penetration, indicating that invasin-mediated entry is the primary chromosomally encoded pathway responsible for Y. pseudotuberculosis penetration into both HEp-2 and Chinese hamster ovary cells under the assay conditions described here.
Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae
Authors: Berry A M , Yother J , Briles D E , Hansman D , Paton J C
Published: January 2020

Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.
Effector cells involved in nonspecific and antibody-dependent mechanisms directed against Plasmodium falciparum blood stages in vitro
Authors: Lunel F , Druilhe P
Published: January 2020

We have evaluated in in vitro conditions the possible cooperative effect of antimalarial antibodies with several human blood cell types. When used alone, immunoglobulin G from African adults who had reached a state of premunition against malaria was found to have no or very limited direct effect on invasion and multiplication of P. falciparum asexual blood stages. In contrast, these antibodies induced a marked specific inhibition of parasite growth in the presence of normal blood monocytes, and the inhibition did not appear to be strain dependent. No similar antibody-dependent cellular inhibitory effect was found using human blood polymorphonuclear leukocytes, lymphocytes, platelets, or adherent spleen cells. However, these cells could all exert in vitro some non-antibody-dependent inhibitory effect when present at high effector/target cell ratios.
Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis
Authors: Hanada N , Kuramitsu H K
Published: January 2020

Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gtfD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gtfD gene in a gtfC gtfB mutant indicated that three distinct gtf genes involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gtf genes from Streptococcus sanguis or Streptococcus sobrinus.
Effect of streptomycin administration on association of enteric pathogens with cecal tissue of mice
Authors: Pongpech P , Hentges D J , Marsh W W , Eberle M E
Published: January 2020

The effect of streptomycin on the ability of Shigella sonnei 3SR and enterotoxigenic Escherichia coli 2SR to associate with cecal tissue of mice in vivo and in vitro was examined. After orogastric challenge, both pathogens associated in significantly greater numbers (P less than or equal to 0.05) with the cecal tissue of streptomycin-treated mice than with the tissue of untreated mice. The population levels of the pathogens were also significantly greater (P less than or equal to 0.05) in the cecal contents of streptomycin-treated mice than in untreated mice. When excised cecal tissues from the two groups of mice were exposed to the pathogens in vitro, the extent of the association of the pathogens was markedly greater with tissues from streptomycin-treated mice than with tissues from untreated mice. There was also a positive correlation between the numbers of the pathogens in the suspending fluid and the extent of the tissue associations. The population size of fusiform organisms, which are the major components of the mucus layer of the ceca of mice, was reduced 100-fold by streptomycin administration. This was determined by microscopic count. Sections of cecal tissue obtained from the mice and stained with hematoxylin and eosin demonstrated that streptomycin administration greatly decreased the number of fusiform bacteria present in the mucosal microbial layer. We speculate that the partial elimination of fusiform organisms from this layer by streptomycin administration provides available association sites for pathogens so that they can successfully colonize the mouse cecum.
Induction of inflammatory mediators (histamine and leukotrienes) from rat peritoneal mast cells and human granulocytes by Pseudomonas aeruginosa strains from burn patients
Authors: Bergmann U , Scheffer J , Köller M , Schönfeld W , Erbs G , Müller F E , König W
Published: January 2020

Clinical isolates of Pseudomonas aeruginosa from severely burned patients were analyzed with regard to their capacity to induce inflammatory-mediator release from rat mast cells or human granulocytes. The bacterial strains were characterized according to their cell-associated hemolysin activity as well as their secreted hemolysin and phospholipase C activities. P. aeruginosa expressing heat-labile hemolysin and phospholipase C induced histamine release from rat mast cells and leukotriene formation from human granulocytes, while bacterial strains expressing heat-stable hemolysin were potent releasers of histamine but did not lead to leukotriene formation. The mediator-inducing capacity was dependent on the growth characteristics of the bacterial strains. The purified glycolipid (heat-stable hemolysin) of P. aeruginosa was a potent inducer of histamine release but did not initiate leukotriene formation. Exotoxin A did not affect inflammatory-mediator release. P. aeruginosa with leukotriene-inducing capacity also enhanced omega oxidation of endogenous leukotriene B4, suggesting an additional inactivation of the chemotactic potential. Our data suggest that both hemolysins of P. aeruginosa contribute to the pathogenicity of P. aeruginosa by inducing and modulating inflammatory-mediator release from various cells.
Relationship between neutrophil-mediated oxidative injury during acute experimental pyelonephritis and chronic renal scarring
Authors: Meylan P R , Markert M , Bille J , Glauser M P
Published: January 2020

Previous experiments with rats have suggested that pyelonephritic scarring after acute ascending Escherichia coli pyelonephritis partly results from excessive polymorphonuclear leukocyte (PMN) infiltration and activation in the kidney parenchyma. We have studied the role of PMN oxidative metabolism in generating tissue injury during acute pyelonephritis. Rats with acute pyelonephritis were treated with dapsone (25 mg/kg twice daily for 3 days), a compound known to prevent PMN oxidant damage. In vitro, levels of dapsone easily achieved in vivo inhibited myeloperoxidase (MPO)-mediated reactions involving the oxidation of halides to reactive cytotoxic hypohalites (such as MPO-mediated iodination and luminol-enhanced chemiluminescence). In contrast, dapsone had no effect on superoxide production, lysosomal enzyme release, or bacterial killing by activated PMN. In vivo, dapsone treatment had no significant effect on acute pyelonephritis with respect to (i) bacterial counts, (ii) inflammatory swelling, and (iii) PMN infiltration. However, dapsone-treated animals sacrificed 2 months after acute pyelonephritis had a 65% reduction of renal scars when compared with controls. Since dapsone had no antibacterial effect, this protection is compatible with the hypothesis that dapsone prevented oxidant-generated tissue injury due to the extracellular release of the MPO system by activated PMN during acute suppurative pyelonephritis.
Colonization of mice by Campylobacter jejuni
Authors: Jesudason M V , Hentges D J , Pongpech P
Published: January 2020

Both streptomycin-treated and untreated Swiss white mice were irregularly colonized when challenged orogastrically with between 1 and 10(11) viable organisms of either of two strains of Campylobacter jejuni. The organisms were occasionally recovered from portions of the intestinal tracts of these animals in numbers ranging from 10(1) to 10(3)/g when the challenge doses were 10(10) or more. When germfree mice were challenged with 10(8) organisms of either strain, the entire intestinal tracts of all the animals were colonized with C. jejuni in numbers ranging from 10(4) to 10(9)/g. The ceca were most heavily colonized. Both strains of C. jejuni multiplied anaerobically in brucella broth, except when the broth contained 60.80 mu eq of total volatile fatty acids (VFA) per ml at pH 6.75, simulating conditions in the ceca of untreated mice, or when it contained 21.63 mu eq/ml at pH 7.04, simulating conditions in the ceca of streptomycin-treated mice. Active multiplication occurred, however, in brucella broth without VFA at pH 7.02 that was incubated microaerobically, simulating conditions in the ceca of germfree mice. The results suggest that VFA operating under anaerobic conditions present in the intestinal tract of both streptomycin-treated and untreated conventional mice interfere with the multiplication of C. jejuni. The organisms actively multiply, on the other hand, in the absence of VFA at the higher oxidation-reduction potential of the intestinal tract of germfree mice.
Cloning and characterization of the structural gene for the class 2 protein of Neisseria meningitidis
Authors: Murakami K , Gotschlich E C , Seiff M E
Published: January 2020

The class 2 protein of Neisseria meningitidis is the major outer membrane protein and a porin. A lambda gt11 bank of meningococcal chromosomal DNA was screened with monoclonal antibodies against gonococcal protein IB that cross-react with meningococcal class 2 protein. Three independent immunoreactive clones were isolated. DNA sequence analysis indicated that these clones included regions encoding the N-terminal portion of the class 2 protein. An oligonucleotide of 21 bases that was complementary to this sequence was synthesized and used as a probe for a second screening of the lambda gt11 bank. One of the positive clones isolated contained the complete gene, including the ribosome binding site, but lacked the promoter region. On the basis of the DNA sequence, a protein of 360 amino acids was predicted. Comparison of the predicted protein sequence with that of gonococcal protein I showed little homology in six regions constituting 29% of the total amino acids, while the remainder of the coding frame showed 81% homology of amino acid residues. The DNA homology in the immediate 5' and 3' noncoding sequences was very striking. Following the putative transcription terminator, the 3' DNA sequence contained a complex pattern of direct and inverted repeats having some similarity to the 3' sequence of the gonococcal protein IB gene and very close homology to a sequence located in the pilS1 region (R. Haas and T. F. Meyer, Cell 44:107, 1986), an area of the gonococcal genome containing silent pilin genes.
Entry of genital Chlamydia trachomatis into polarized human epithelial cells
Authors: Wyrick P B , Choong J , Davis C H , Knight S T , Royal M O , Maslow A S , Bagnell C R
Published: January 2020

To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A.S. Maslow, C.H. Davis, J. Choong, and P.B. Wyrick, Am. J. Obstet. Gynecol. 159:1006-1014, 1988). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens.
Ultrastructural localization of the fibrinogen-binding domain of streptococcal M protein
Authors: Rýc M , Beachey E H , Whitnack E
Published: January 2020

Binding of fibrinogen to the M protein located on the surface fibrillae of group A streptococci impedes deposition of complement and thus contributes to the virulence of these organisms. We investigated this binding by electron microscopy using postembedding immunogold labeling. Both fibrinogen and its D fragment formed a distinct dense layer in the surface fibrillae, separated by 10 nm from the compact part of the cell wall. Labeling the sections with anti-fibrinogen or anti-fragment D showed that the fibrinogen-binding region lay within a 25-nm segment of the fibrillae beginning approximately 30 nm from the inner surface of the cell wall. The outer surface of the fibrinogen layer could be labeled with antibody to the amino-terminal half of type 24 M protein, indicating that the fibrillar tips remained exposed after fibrinogen binding. The degree of labeling with anti-fibrinogen, determined by gold particle counting, was the same whether the bacterial cells had been incubated with purified fibrinogen or whole plasma. These results indicate that the fibrinogen-binding region lies in the distal (amino-terminal) half of the M protein molecule but excludes the most distal portion, which is the site of epitopes that interact with opsonic anti-M antibody, and that plasma proteins other than fibrinogen, a number of which are known to bind to group A streptococci, do not interfere with fibrinogen binding.
Selective inhibition of cytokine-induced lysozyme activity by tetanus toxin in the GG2EE macrophage cell line
Authors: Pitzurra L , Marconi P , Bistoni F , Blasi E
Published: January 2020

This study was designed to evaluate the effects of tetanus toxin (TT) on lysozyme (LZM) activity by the GG2EE macrophage cell line. GG2EE cells spontaneously produced low amounts of LZM, which were mostly secreted into the culture medium. Upon treatment with various cytokines, GG2EE cells exhibited altered LZM activity. In particular, exposure of GG2EE cells to alpha/beta interferon (IFN-alpha/beta) reduced LZM activity, as opposed to treatment with gamma interferon (IFN-gamma) or colony-stimulating factor 1, which potentiated LZM activity. Spontaneous LZM activity of GG2EE cells was not susceptible to TT action; in contrast, when IFN-gamma- or colony-stimulating factor 1-susceptible cells were treated with TT, a significant reduction on LZM activity was observed. The TT inhibitory effect was dose dependent and manifested only after a 6-h incubation of GG2EE cells with TT. Treatment of GG2EE cells with heat-inactivated TT as well as Ibc- and B-IIb-TT-derived fragments was found to be ineffective, while pretreatment with B-IIb- but not with Ibc-TT-derived fragment abrogated the TT effect. Overall, these data indicate the existence of a specific TT-GG2EE cell interaction, leading to selective inhibition of cytokine-induced LZM activity.
Purification of an 80,000-Mr glycylprolyl peptidase from Bacteroides gingivalis
Authors: Barua P K , Neiders M E , Topolnycky A , Zambon J J , Birkedal-Hansen H
Published: January 2020

An enzyme from Bacteroides gingivalis SUNYAB A7A1-28 that hydrolyzes the synthetic peptide glycyl-L-proline 4-methoxy-beta-naphthylamide was purified 1,040-fold by urea extraction, gel filtration, ion-exchange chromatography, and fast protein liquid chromatography. The molecular weight of the enzyme was 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 75,000 as determined by gel filtration. The optimum pH for the hydrolysis of glycyl-L-proline 4-methoxy-beta-naphthylamide was 7.5 to 8.5. The enzyme activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride by 82.5 and 78%, respectively. The activity was also inhibited by Hg2+ (55.6%) and Zn2+ (45%).
Haemophilus influenzae outer membrane vesicle-induced blood-brain barrier permeability during experimental meningitis
Authors: Wispelwey B , Hansen E J , Scheld W M
Published: January 2020

Haemophilus influenzae type b (Hib) lipopolysaccharide (LPS) may be present in the cerebrospinal fluid largely as part of outer membrane vesicles (OMV), which could possibly alter its activity. Similar to inoculation of purified Hib LPS, intracisternal inoculation of Hib OMV into adult rats resulted in dose- and time-dependent increases in blood-brain barrier permeability. Polymyxin B, but not an oligosaccharide-specific monoclonal antibody, significantly inhibited the activity of Hib OMV. No change in blood-brain barrier permeability occurred in leukopenic rats inoculated with Hib OMV. Hib OMV was as active as purified Hib LPS on a weight basis and therefore appears to be a relevant vehicle for the delivery of LPS during meningitis.
Mapping of the human plasmin domain recognized by the unique plasmin receptor of group A streptococci
Authors: Broder C C , Lottenberg R , Boyle M D
Published: January 2020

A high-affinity surface receptor for human plasmin has been reported on certain group A streptococci. To map the region of the plasmin molecule that binds to the bacterial receptor, isolated domains of plasmin were tested for their ability to inhibit the binding of intact radiolabeled plasmin to receptor-positive bacteria. Complete inhibition of binding of labeled plasmin to bacteria by isolated heavy chains was achieved, but this inhibition was not as efficient on a molar basis when compared with that of unlabeled plasmin. By contrast, a conformationally altered form of native plasminogen was found to bind to bacteria and was as efficient a competitive inhibitor as intact plasmin was. The results of this study indicate that the selective binding of human plasmin to a group A streptococcus is dependent on structures present in the conformationally altered form of native plasminogen or plasmin that are not found on the native zymogen, the plasminogen with NH2-terminal glutamic acid.
Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae
Authors: Tsai W M , Larsen S H , Wilde C E
Published: January 2020

The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.
Activity of polymorphonuclear leukocytes in the presence of sulfide
Authors: Claesson R , Granlund-Edstedt M , Persson S , Carlsson J
Published: January 2020

Polymorphonuclear leukocytes (PMN) isolated from human blood were exposed to various levels of hydrogen sulfide. The effect on respiratory burst, myeloperoxidase activity, and capacity to phagocytose and kill bacteria were studied. A 1-h exposure of the PMN to 1 mM sulfide did not decrease their myeloperoxidase activity or their capacity to initiate a respiratory burst. Actually the products of the respiratory burst rapidly oxidized sulfide. The phagocytosis and killing of bacteria in the presence of 1 mM sulfide was only decreased to a minor extent. Myeloperoxidase in cell extract was, however, almost completely inhibited by 1 microM sulfide. These results indicate that hydrogen sulfide does not easily permeate PMN. PMN may be able to function in infected sites with high sulfide levels such as in the gingival pockets of periodontal disease. In the oxygenated areas of these sites the PMN may actually help in the detoxification of sulfide.
Macrophage cell lines P388D1 and IC-21 stimulated with gamma interferon fail to inhibit the intracellular growth of Histoplasma capsulatum
Authors: Wu-Hsieh B , Howard D H
Published: January 2020

Histoplasma capsulatum, a facultative intracellular parasite of macrophages, grows within mononuclear cells of the P388D1 and IC-21 cell lines with a generation time comparable to that with which it grows in normal resident peritoneal macrophages (10 +/- 2 h). Recombinant murine gamma interferon (rMuIFN-gamma) activates P388D1 cells to express la antigens but not to inhibit the intracellular growth of H. capsulatum, alone or in combination with lipopolysaccharide. IC-21 cells also could not be activated to fungistasis with rMuIFN-gamma. Explanted resident peritoneal macrophages of the C57BL/6 (from which the IC-21 cell line derives), C3H/HeJ, DBA/2 (from which the P388D1 cell line derives), A/J, and SJL/J strains of mice were all stimulated by rMuIFN-gamma to inhibit the fungus.