bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine natural killer cells are fungicidal to Cryptococcus neoformans
Authors: Hidore M R , Nabavi N , Sonleitner F , Murphy J W
Published: January 2020

Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putorius furo)
Authors: Fox J G , Otto G , Taylor N S , Rosenblad W , Murphy J C
Published: January 2020

Helicobacter mustelae has been cultured from the stomachs of ferrets with chronic gastritis; the lesions in the stomach have many of the same histological features seen in H. pylori gastritis in humans. To determine whether H. mustelae-negative ferrets with normal gastric mucosa were susceptible to colonization and whether gastritis developed after infection, four H. mustelae-negative ferrets treated with cimetidine were inoculated orally on two successive days with 3 ml (1.5 x 10(8) CFU) of H. mustelae; eight age-matched H. mustelae-negative ferrets served as controls. All four ferrets became colonized; H. mustelae persisted through week 24 of the study, as determined by positive gastric culture, tissue urease, and Warthin-Starry staining of gastric tissue. Superficial gastritis developed in the oxyntic gastric mucosa, and a full-thickness gastritis, composed primarily of lymphocytes and plasma cells plus small numbers of neutrophils and eosinophils, was present in the antrum. The inflammation was accompanied by an elevation of immunoglobulin G antibody to H. mustelae. At 4 weeks post-inoculation, the four infected (experimental) ferrets developed an elevated gastric pH (4.0 to 5.2) for 2 weeks. The eight control ferrets did not have gastritis; H. mustelae could not be demonstrated in gastric tissue via culture, nor was there an immune response to the bacteria. In ferrets, H. mustelae readily colonizes the stomach and produces a gastritis, a significant immune response, and, like H. pylori infection in humans, a transient elevated gastric pH after Helicobacter infection.
Local skin response in mice induced by a single intradermal injection of bacterial lipopolysaccharide and lipid A
Authors: Ishikawa Y , Kirikae T , Hirata M , Yoshida M , Haishima Y , Kondo S , Hisatsune K
Published: January 2020

Dermal inflammation and hemorrhagic necrosis induced by bacterial lipopolysaccharide (LPS) and lipid A were studied in mice. In ddY mice, a single intradermal injection of Salmonella typhimurium S-form LPS and lipid A into the abdominal dermis elicited an edematous change due to an increase in local vascular permeability 12 h postinjection, followed by hemorrhagic necrosis from 24 to 72 h. This skin reaction was also induced in a dose-dependent manner by S-form LPS, R-mutant LPS, and lipid A of S. typhimurium and Escherichia coli, but not by polysaccharide from Salmonella S-form LPS. The dermal inflammation-inducing activities of LPS and lipid A were roughly in the following order (from highest to lowest): Re-form LPS, Rc-form LPS and lipid A, Ra-form LPS, and S-form LPS. These results suggest that the lipid A portion of the LPS molecule is responsible for the skin reaction. In C3H/HeN mice, Re-form LPS and lipid A induced the same intensity of skin reaction as that in ddY mice. In C3H/HeJ mice, which have a low response to LPS, Re-LPS and lipid A did not induce any hemorrhagic response but showed a distinct edematous change. Although hemorrhagic necrosis and edematous changes could be explained by quantitative differences in skin lesions, the other possible explanation is that hemorrhagic necrosis and the increase in local vascular permeability are induced by different mechanisms, only one of which depends on the regulation of the lps gene.
Bacteroides (Porphyromonas) gingivalis fimbriae activate mouse peritoneal macrophages and induce gene expression and production of interleukin-1
Authors: Hanazawa S , Murakami Y , Hirose K , Amano S , Ohmori Y , Higuchi H , Kitano S
Published: January 2020

The purpose of this study was to examine whether Bacteroides (Porphyromonas) gingivalis fimbriae, an important structure involved in attachment of the bacteria to periodontal tissues, activate macrophages and subsequently induce gene expression and production of interleukin-1 (IL-1) in the cells. The fimbriae increased glucose consumption and lysozyme activity in BALB/c macrophages, both criteria of macrophage activation of peritoneal macrophages, in a dose-dependent fashion. A marked increase in the mRNA level of the c-myc gene, an oncogene, in the cells was observed after a 1-h treatment with the fimbriae, and the level decreased rapidly after 3 h. The fimbriae (4 micrograms of protein per ml) markedly induced IL-1 alpha and IL-1 beta gene expression in the cells and IL-1 production. The expression of IL-1 alpha and IL-1 beta genes measured in terms of specific mRNA increased 1 h after the start of treatment and peaked at 6 h. Such increased expression of IL-1 beta was also observed in C3H/HeJ mice, a lipopolysaccharide low-responder strain. The fimbriae stimulated transcriptional activity of IL-1 beta in the cells, but not that of IL-1 alpha. We also observed that fimbriae-induced IL-1 gene expression was not regulated by endogenous prostaglandin triggered by the fimbriae. Therefore, these observations suggest that B. gingivalis fimbriae may be involved in the pathogenesis of adult periodontal disease via triggering of IL-1 production by monocytes/macrophages in periodontal diseases.
Modulation of experimental autoimmunity: treatment of adjuvant arthritis by immunization with a recombinant vaccinia virus
Authors: Hogervorst E J , Schouls L , Wagenaar J P , Boog C J , Spaan W J , van Embden J D , van Eden W
Published: January 2020

Live recombinant vaccinia viruses, expressing antigens from pathogenic microorganisms, are studied for their use as vaccines designed for the protection against infectious diseases. Infections with these vaccinia virus recombinants, expressing proteins or epitopes from viruses, parasites, or bacteria, have resulted in the development of specific neutralizing antibodies or cytotoxic T lymphocytes. Here, we describe the generation of a recombinant vaccinia virus expressing the mycobacterial 65-kDa heat shock protein (HSP65). A vaccinia recombinant virus was constructed by placing the gene for the Mycobacterium bovis BCG HSP65 under control of a vaccinia virus promoter and inserting this mycobacterial gene in the thymidine kinase locus of the vaccinia virus genome. Mycobacterial HSP65 is a critical antigen in the autoimmune model of adjuvant arthritis induced in Lewis rats by the immunization with Mycobacterium tuberculosis. We report the induction of immunity directed to this mycobacterial HSP65 by testing for the presence of specific antibodies and T-cell proliferation. Furthermore, induction of such immunity resulted in a reduction of arthritis severity when given to rats before or, even more interestingly, during development of arthritis. Disease reduction was not found after administration of HSP65 in the absence of vaccinia virus as a vector when given during arthritis development. Therefore, recombinant vaccinia virus may offer new prospectives for specific intervention in autoimmunity.
Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis
Authors: Pantosti A , Tzianabos A O , Onderdonk A B , Kasper D L
Published: January 2020

Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physiochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans.
Antibody-dependent alternate pathway of complement activation in opsonophagocytosis of Porphyromonas gingivalis
Authors: Cutler C W , Kalmar J R , Arnold R R
Published: January 2020

It has been suggested that the ability of Porphyromonas gingivalis to proteolyse complement, as well as its production of a capsule, contributes to resistance to phagocytosis by polymorphonuclear leukocytes. In this report, the opsonic role of serum complement and its activation pathways were investigated, using individual sera heat treated or depleted of factors B, C2, and C1q and the divalent cations Mg2+ and Ca2+. A fluorochrome microassay was used to quantitate phagocytosis of P. gingivalis A7436 by human polymorphonuclear leukocytes. Heat treatment of rabbit antiserum to P. gingivalis (RaPg) (56 degrees C, 30 min) resulted in a reduction in phagocytosis from 100% to 55% +/- 5%, while heat treatment of chronic adult periodontal disease serum abrogated phagocytosis. The heat-labile activity of RaPg was fully restored with MgEGTA-chelated rabbit serum but not EDTA- or EGTA-chelated rabbit serum. The addition of serum depleted of factor B but not C2 or C1q restored most of the heat-labile activity; however, the factor B-depleted serum was suspect, due to low-level opsonization of zymosan (inhibitable by EDTA but not MgEGTA). Adding C1q at 80 micrograms/ml to serum depleted of C1q restored much but not all of the activity lost through heat treatment or through depletion of C1q. A large part of opsonic activity with C2- and C1q-depleted sera was enhanced by the addition of 4 x 10(-3) M Mg2+. The data indicate that although opsonophagocytosis of P. gingivalis A7436 is dependent on the classical complement pathway, a significant contribution is made by an antibody-dependent alternate pathway.
Tumor necrosis factor alpha mediates lethal activity of killed gram-negative and gram-positive bacteria in D-galactosamine-treated mice
Authors: Freudenberg M A , Galanos C
Published: January 2020

Treatment with D-galactosamine increases sensitivity of lipopolysaccharide (LPS)-responder mice to the lethal effects of LPS, while nonresponder mice remain resistant (M.A. Freudenberg, D. Keppler, and C. Galanos, Infect. Immun. 51:891-895, 1986). In the present study it is shown that, in contrast to LPS, killed gram-negative bacteria (Salmonella abortus equi and S. typhimurium) were highly toxic for D-galactosamine-treated LPS-responder (C57BL/10 ScSN and C3H/HeN) and -nonresponder (C57BL/10 ScCR and C3H/HeJ) mice, although to a higher extent in the former strains. Also, killed gram-positive bacteria (Staphylococcus aureus, Propionibacterium acnes, and Mycobacterium phlei) exhibited toxicity for D-galactosamine-treated mice, LPS-responder and -nonresponder mice being equally susceptible. Evidently, bacterial components other than LPS may exhibit lethal effects in sensitized animals. In all cases, the lethality of LPS and of bacteria was inhibited by anti-tumor necrosis factor alpha (TNF-alpha) serum. While LPS induced TNF-alpha in vitro only in macrophages from LPS-responder mice, gram-negative and gram-positive bacteria induced TNF-alpha also in macrophages from LPS-nonresponder mice. The data show that TNF-alpha is a common endogenous mediator of the lethal activity of gram-negative and gram-positive bacteria.
Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes
Authors: Siefferman C M , Regelmann W E , Gray B H
Published: January 2020

The susceptibility of paired mucoid and nonmucoid variants of Pseudomonas aeruginosa isolated from 13 patients with cystic fibrosis (CF) to killing by a 55,000-Da bactericidal protein (BP55) from human polymorphonuclear leukocytes was studied. Mucoid and nonmucoid variants were equally sensitive to killing by BP55 at both pH 5.6 and pH 7.2. Eleven of the isolates were resistant to the bactericidal activity of 10% normal human serum but were as sensitive as the serum-sensitive isolates to BP55. Similarly, the 15 isolates with lipopolysaccharides (LPS) containing O-polysaccharide side chains (smooth LPS) were as sensitive to BP55 as those isolates with rough LPS.P. aeruginosa isolates from patients in poor clinical condition were more likely to have LPS of the smooth type and to be resistant to killing by 10% human serum than the isolates from patients in good clinical condition. We have concluded that the susceptibility of the P. aeruginosa isolates from patients with CF to killing by BP55 does not correlate with mucoid or nonmucoid variations, with the presence or absence of smooth LPS, or with the sensitivity or resistance to killing by normal human serum.
Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin
Authors: Newton S M , Kotb M , Poirier T P , Stocker B A , Beachey E H
Published: January 2020

A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.
A 220-kilodalton glycoprotein in yeast extract inhibits Staphylococcus aureus adherence to human endothelial cells
Authors: Elliott D A , Hatcher V B , Lowy F D
Published: January 2020

A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.
Elimination of hydrogen peroxide by Haemophilus somnus, a catalase-negative pathogen of cattle
Authors: Sample A K , Czuprynski C J
Published: January 2020

Haemophilus somnus is a catalase-negative, gram-negative pathogen of cattle which is refractory to killing by bovine neutrophils. In this report, we showed that H. somnus rapidly inhibited Luminol-dependent chemiluminescence of bovine neutrophils costimulated with opsonized zymosan or phorbol myristate acetate. We have postulated that this inhibition resulted in part from H. somnus preventing the accumulation of hydrogen peroxide (H2O2) during the oxidative burst. In support of this hypothesis, we have demonstrated that when stimulated with viable H. somnus, bovine neutrophils accumulate lower levels of H2O2 than did neutrophils stimulated with heat-killed H. somnus or opsonized zymosan. We have presented evidence that four separate strains of H. somnus, despite being catalase negative by conventional criteria, removed H2O2 from solution. Viable cells of H. somnus were required for the removal of H2O2 from solution; little or no activity was observed when suspensions of heat-killed, formalin-killed, or sonicated cells of H. somnus were incubated with H2O2. In addition, the elimination of H2O2 occurred only in the presence of carbon sources that could be utilized by H. somnus, indicating that elimination of H2O2 was an energy-dependent process. The amount of H2O2 that could be eliminated by 10(7) cells of H. somnus was greater than 10 nmol, an amount comparable to that produced by a similar number of stimulated bovine neutrophils. Thus, we suggest that the ability of H. somnus to remove H2O2 from solution may be an important virulence mechanism that contributes to the survival of the organism following ingestion by bovine neutrophils.
An immunoreactive apoglycoprotein purified from Coccidioides immitis
Authors: Dugger K O , Galgiani J N , Ampel N M , Sun S H , Magee D M , Harrison J , Law J H
Published: January 2020

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and greater than 200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.
Acquisition of iron by Legionella pneumophila: role of iron reductase
Authors: Johnson W , Varner L , Poch M
Published: January 2020

Legionella pneumophila has been shown to survive and multiply in a variety of intracellular environments, including protozoa and human mononuclear phagocytes. However, the mechanism by which this organism acquires iron in the intracellular environment has not been studied. Since L. pneumophila does not produce siderophores, alternative methods of iron acquisition were investigated. Virulent strains of L. pneumophila were able to grow in media containing as little as 3 microM iron, whereas avirulent cells required a minimum of 13 microM iron for growth. Neither virulent nor avirulent cells were able to utilize 55Fe bound to transferrin. When incubated in the presence of 55Fe in the form of ferric chloride, both virulent and avirulent cells accumulated equal amounts of iron. The uptake of iron was energy dependent as indicated by inhibition of 55Fe uptake at 4 degrees C and preincubation of the cells with KCN. Treatment of virulent cells with pronase or trypsin had no effect on iron uptake. In contrast, pronase or trypsin treatment of avirulent cells resulted in increased uptake of iron. Iron reductase activity in both virulent and avirulent cells was demonstrated, with the highest specific activity associated with the periplasmic fraction. Maximum reductase activity of virulent cells occurred with NADH as the reductant. In contrast, avirulent cells showed a twofold increase in enzyme activity when NADPH was used as the reductant. These results suggest that an iron reductase is important in iron acquisition by L. pneumophila.
Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes
Authors: Geoffroy C , Raveneau J , Beretti J L , Lecroisey A , Vazquez-Boland J A , Alouf J E , Berche P
Published: January 2020

We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. welshimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established.
Lipopolysaccharidelike immunological properties of cell wall glycoproteins isolated from Cytophaga johnsonae
Authors: de Jong D M , Pate J L , Kirkland T N , Taylor C E , Baker P J , Takayama K
Published: January 2020

Glycoproteins (GP) previously shown to be involved in the gliding motility of Cytophaga johnsonae were examined for biological activities characteristic of lipopolysaccharide (LPS). These integral membrane proteins activated 70Z/3 pre-B cells to synthesize immunoglobulin M, induced B cells to synthesize non-antigen-specific polyclonal immunoglobulin, induced macrophages to produce tumor necrosis factor, and modulated the antibody response to type III pneumococcal polysaccharide in the absence of thymus-derived (T) lymphocytes. Except for the GP activity in the 70Z/3 assay, all activities of the GP were comparable to or greater than those of LPS. No LPS was detected in the preparations of GP used or in the phenol-water extracts of C. johnsonae. The mechanism by which these GP exerted their biological activities was distinct from that of LPS, since LPS-resistant C3H/HeJ mice responded to GP. Furthermore, biologically inactive diphosphoryl lipid A obtained from nontoxic LPS of Rhodopseudomonas sphaeroides (an analog of toxic lipid A), which is an antagonist of LPS, did not block the induction of tumor necrosis factor by GP in macrophages. These results showed that the cell surface GP from C. johnsonae are potent LPS-like activators of B cells and macrophages. We suggest that these GP might be good candidates for use in developing an effective adjuvant system.
Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains
Authors: Hermans P W , van Soolingen D , Bik E M , de Haas P E , Dale J W , van Embden J D
Published: January 2020

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.
Virulence of Wolinella recta in a murine abscess model
Authors: Kesavalu L , Holt S C , Crawley R R , Borinski R , Ebersole J L
Published: January 2020

The virulence of Wolinella recta isolates was studied in an experimental animal model by using monoinfection of BALB/c mice. Infection with clinical isolates of W. recta 576 and W. recta 234 induced dry, flat, depressed gangrenous necrotic skin lesions, whereas W. recta ATCC 33238 failed to induce a similar lesion. Histological examination of the skin lesion 72 h postinfection revealed coagulation necrosis of the epidermis, subcutis and cutaneous truncus muscle, with marked exudation of serum proteins and neutrophils. Virulence-modulating agents such as dexamethasone, galactosamine, hydrazine sulfate, and dextran microcarrier beads were used in conjunction with W. recta infection. Dexamethasone, hydrazine sulfate, and dextran beads enhanced the infectivity and pathogenicity of W. recta for lesion formation and tissue destruction compared with what was found in untreated control mice. Galactosamine sensitization enhanced the virulence potential of W. recta to such an extent that a lethal outcome was observed. Laboratory passage of clinical isolates demonstrated a decreased virulence in high-passage strains, which correlated with the minimal virulence observed in the extensively passaged W. recta ATCC 33238. Serum immunoglobulin G (IgG) and IgM responses were detected in the serum of infected animals, and cross-reacting antibody indicated variation in the antigenic makeup of various W. recta strains. Enhanced IgG antibody responses were observed following the secondary challenge. Mice with acquired antibody response to initial infection remained susceptible to lesion formation with subsequent challenge, but the size of the lesion was significantly reduced, indicating partial protection. Serum IgG and IgM antibody levels were significantly increased by active immunization when compared with levels in mice which had recovered from infection. The immunization significantly decreased the lesion size; however, even these high levels of antibody failed to abrogate the lesion induction.
Persistence of Escherichia coli bacteriuria is not determined by bacterial adherence
Authors: Andersson P , Engberg I , Lidin-Janson G , Lincoln K , Hull R , Hull S , Svanborg C
Published: January 2020

The role of bacterial adherence in the persistence of bacteria in the human urinary tract was analyzed. Women with chronic symptomatic urinary tract infections were subjected to deliberate colonization with nonvirulent Escherichia coli, after eradication of their current infections. E. coli organisms were instilled into the bladder through a catheter. The strain used for colonization, E. coli 83972, was isolated from a patient with stable bacteriuria. It lacked expressed adherence factors and did not belong to the uropathogenic O:K:H serotypes. Strain 83972 was transformed with the pap and pil DNA sequences encoding Gal alpha 1-4Gal beta- and mannose-specific (type 1) adhesins. Patients were colonized with a mixture of the wild-type and the transformed strains. E. coli 83972 caused stable bacteriuria for greater than 30 days in 7 of 12 individuals. In contrast, the Gal alpha 1-4 Gal beta-recognizing or mannose-binding transformants were eliminated within 48 h. The consistent superiority of the wild-type strain in establishing stable bacteriuria when compared with the adhesive transformants did not appear to be due to differences in growth rates or to plasmid segregation. Rather, the transformants expressing the adhesin determinants were selectively eliminated by the host. This suggested that the acquisition of adherence factors is not sufficient to increase the fitness of E. coli for survival in the urinary tract of humans.
Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin
Authors: Bhakdi S , Martin E
Published: January 2020

Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients. The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids. The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation. When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of superoxide anions; shortly thereafter, irreversible membrane permeabilization occurred. When the toxin was applied at concentrations exceeding 0.2 to 0.3 HU/ml, membrane permeabilization was so rapid that the cells were unable to mount a respiratory burst. When applied in the narrow range of 0.05 to 0.1 HU/ml, E. coli hemolysin rivaled phorbol myristate acetate in its capacity to stimulate production of superoxide anions. Additionally, hemolysin applied at doses that elicited no pore formation (0.01 to 0.02 HU/ml) primed leukocytes for an augmented response to subsequent challenge by the phorbol ester. These data demonstrate that very low doses of E. coli hemolysin can evoke cellular reactions that appear independent of and precede transmembrane pore formation and cell death.
Topology of outer membrane porins in pathogenic Neisseria spp
Authors: van der Ley P , Heckels J E , Virji M , Hoogerhout P , Poolman J T
Published: January 2020

In Escherichia coli, membrane-spanning amphipathic beta-sheet structures are characteristic of many outer membrane proteins. By applying the principles that have been recognized for them to the four classes of neisserial porins, we have constructed a model for the topology of the porins within the outer membrane. This model predicts eight surface-exposed loops, both in the meningococcal class 1 and 2 proteins and in the gonococcal PIA and PIB proteins. The transmembrane sequences are highly conserved among these porins and are able to form an amphipathic beta-sheet structure. The surface-exposed hydrophilic loops show extensive variation in both length and sequence. Experimental evidence in support of this model has been obtained by using antisera against synthetic peptides which correspond to surface-exposed loops in class 1 and 2 proteins. Thus, binding to the cell surface was observed with antibodies against loops 1, 4, and 5 of class 1 and loops 1 and 5 of class 2. In class 1, these loops are the longest ones and show the highest sequence diversity among strains of different subtypes. Mapping of epitopes recognized by monoclonal antibodies with bactericidal activity has also provided strong support for the model. The epitopes are located in loops 1 and 4 of class 1 protein, loop 5 of PIB, and loop 6 of PIA. A nonbactericidal antibody that binds only weakly to whole cells was shown to recognize loop 3 of PIB. These results suggest that the longest loops are immunodominant, provide the binding sites for bactericidal antibodies, and display the greatest variation among different strains.
Live but not heat-killed mycobacteria cause rapid chemotaxis of large numbers of eosinophils in vivo and are ingested by the attracted granulocytes
Authors: Castro A G , Esaguy N , Macedo P M , Aguas A P , Silva M T
Published: January 2020

We studied leukocyte chemotaxis triggered by a local injection of mycobacteria (Mycobacterium avium and M. smegmatis) in BALB/c and C57BL/6 mice. Our experimental model consisted of the induction of a subcutaneous air pouch in the dorsal area of mice and inoculation 6 days later of 10(8) CFU of myocobacteria. Inflammatory exudates were harvested from the air pouch cavities 15, 30, and 45 min after the injection of the inocula. Injection of the microorganisms resulted in the migration of an elevated number of eosinophilic granulocytes into the inflammatory cavities. At 30 min after the inoculation of the mycobacteria, the air pouches contained between (3.9 +/- 0.3) x 10(5) (M. avium) and (3.3 +/- 0.3) x 10(5) (M. smegmatis) eosinophils, corresponding to more than one-third (41.4 to 38.3%) of the leukocytes present in the inflammatory cavities. Less than one-half of the eosinophils were attracted to the air pouches when the same number of heat-killed mycobacteria were inoculated [(1.3 +/- 0.2) x 10(5) cells for M. avium and (1.5 +/- 0.2) x 10(5) cells for M. smegmatis]. Injection of gram-negative bacteria (Escherichia coli), of latex beads, or of casein resulted in the attraction of inflammatory eosinophils in numbers that were comparable to those attracted by the heat-killed mycobacteria. Our data document the fact that live mycobacteria exert a rapid chemotactic effect on eosinophils. We therefore postulate that mycobacteria either contain or induce the production of an eosinophilotactic factor. Because this chemotactic effect occurs during the acute inflammatory response to mycobacteria, it cannot be due to the formation of immune complexes (a major infection-associated chemotactic factor for eosinophils). The attracted eosinophils had an important role in the local phagocytosis of mycobacteria, as indicated by our finding, derived from thin-section electron microscopy quantifications, that at 30 min after M. avium inoculation the inflammatory exudates contained (2.2 +/- 0.5) x 10(5) mycobacterium-bearing eosinophils (corresponding to 57% of the total eosinophils), as compared with (2.1 +/- 0.1) x 10(5) neutrophils and (1.5 +/- 0.2) x 10(5) macrophages with ingested bacilli. We conclude that mycobacteria induce the attraction of eosinophils to inflammatory sites and that these granulocytes have the capacity to phagocytize these bacilli in situ.
Capsulelike surface material of Mycoplasma dispar induced by in vitro growth in culture with bovine cells is antigenically related to similar structures expressed in vivo
Authors: Almeida R A , Rosenbusch R F
Published: January 2020

Electron microscopy has been used to show that Mycoplasma dispar produces an external capsulelike material in vivo that has an affinity for both ruthenium red and polycationic ferritin. This extracellular material is lost upon passage in culture medium but can be regained with a single passage on bovine lung fibroblast (BLF) cells. To confirm that the extracellular material associated with cell-grown mycoplasmas was the same as that observed in infected calves, rabbit antibodies were produced to purified capsulelike material isolated by protease digestion of cell-grown organisms. These antibodies bound to capsulelike material on the surface of M. dispar cells colonizing the bronchial epithelium of infected calves and to capsulelike material from cell-grown mycoplasmas. Calves infected with M. dispar produced antibodies in lung secretions that were capable of binding to the purified capsulelike material. The Fab fragments of rabbit antibodies to in vitro-produced capsulelike material could block this binding, indicating that the capsulelike material was similar in both in vivo-grown and cell-grown organisms. The carbohydrate nature of the capsular material suggested by the ruthenium red and polycationic staining characteristics was confirmed by its binding to Ricinus communis agglutinin, a galactose-specific lectin. These studies confirm that capsule material produced during infections with M. dispar share antigenic determinants with the material produced under in vitro conditions and that association with mammalian cells induces production of this material.
Depolarization of polymorphonuclear leukocytes by Porphyromonas (Bacteroides) gingivalis 381 in the absence of respiratory burst activation
Authors: Novak M J , Cohen H J
Published: January 2020

Bacteroides spp. may contribute to the chronicity of mixed infections by affecting the normal functions of polymorphonuclear leukocytes (PMN). This study evaluated the physiologic and biochemical responses of human peripheral blood PMN to a variety of strains of the oral periodontal pathogen Porphyromonas (Bacteroides) gingivalis. Strain 381 and ATCC type strain 33277 caused rapid and lasting depolarization of the electrochemical potential that exists across the PMN membrane by a mechanism that was independent of activation of the pertussis toxin-sensitive G protein or protein kinase C. Membrane depolarization did not initiate increases in intracellular calcium or respiratory burst activation, and activity was not inhibited by surface proteolysis or sugars. However, membrane depolarization was associated with inhibition of PMN responses to the chemotactic peptide N-formylmethionyl leucyl phenylalanine. Membrane-depolarizing activity was isolated with the outer membrane of strain 381 by surface extraction of the bacteria by using Zwittergent 3,14, followed by Sephacryl S-200 gel filtration chromatography. The partially purified outer membrane components were heat stable, were not inhibited by tosyl-lysine chloromethyl ketone, and inhibited N-formylmethionyl leucyl phenylalanine-stimulated superoxide production. The results suggest that outer membrane components of P. gingivalis 381 and 33277 have porinlike activity that can depolarize PMN membranes and immobilize PMN responses to chemotactic peptides. This may prove to be an important virulence characteristic of these strains.
Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses
Authors: Di Tommaso A , Domenighini M , Bugnoli M , Tagliabue A , Rappuoli R , De Magistris M T
Published: January 2020

Filamentous hemagglutinin (FHA), a 220-kDa protein that mediates the adhesion of Bordetella pertussis to eukaryotic cells, is a component of acellular vaccines against whooping cough. To identify the subregions of FHA that are immunogenic for T cells, 16 human T-cell clones were raised against purified FHA and tested for the recognition of recombinant and proteolytic fragments. The clones were found to map either in the carboxy-terminal or the amino-terminal part of the FHA molecule, but none of them recognized the central region, which contains a sequence that is homologous to that of the eukaryotic protein fibronectin. These data suggest that subregions of FHA that do not contain sequences that are potentially cross-reactive with self proteins may be sufficient to induce an immune response against the whole protein.
Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives
Authors: Muller M , Sorrell T C
Published: January 2020

Pyocyanin, a phenazine pigment produced by Pseudomonas aeruginosa, and its metabolite 1-hydroxyphenazine inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid production by up to 70% in human neutrophils stimulated with the calcium ionophore A23187 (5 microM). This potential anti-inflammatory effect was dose dependent and occurred at low concentrations (10 to 50 microM) that did not inhibit neutrophil viability.
Specific Macrophage Immunity to Vaccinia Virus: Macrophage-Virus Interaction
Authors: Avila F. R. , Schultz R. M. , Tompkins W. A. F.
Published: January 2020

Macrophage immunity to vaccinia virus was examined by measuring replication of vaccinia virus in immune and nonimmune rabbit peritoneal macrophages. Vaccinia virus multiplied in macrophages from normal rabbits but failed to replicate in macrophages from immune rabbits. The specificity of vaccinia restriction was demonstrated by the ability of myxoma virus to replicate in nonimmune and vaccinia-immune macrophages. Vaccinia virus absorbed equally well to immune and nonimmune macrophages, but only a small fraction of immune cells supported synthesis of virus antigen. Virus eclipse and infective center neutralization studies suggested that much of the virus remained at the plasma membrane of immune macrophages, even after prolonged incubation in culture. These data support our previous suggestions that macrophages may contribute to specific immunity against vaccinia virus.
Effect of Anti-B Antiserum on the Phagocytosis of
Escherichia coli
Authors: Check Jerome H. , O'Neill Elizabeth A. , O'Neill Karen E. , Fuscaldo Kathryn E.
Published: January 2020

An increase in phagocytosis of
Escherichia coli
O86, an organism containing blood group B substance, was seen in the presence of anti-B antibodies.
Effect of Endotoxin on the Activation of Phosphoenolpyruvate Carboxykinase by Tryptophan
Authors: Rippe Delfin F. , Berry L. Joe
Published: January 2020

The effect of endotoxin on the activation of phosphoenolpyruvate carboxykinase by tryptophan was studied.
Classification of
Neisseria meningitidis
Group B into Distinct Serotypes II. Extraction of Type-Specific Antigens for Serotyping by Precipitin Techniques
Authors: Frasch Carl E. , Chapman S. Stephen
Published: January 2020

Over 10 distinct serotypes of group B
Neisseria meningitidis
have been found to date by using a sensitive microbactericidal assay developed by the authors. The serotype antigens have now been extracted by hot acid or saline extraction procedures. It was found that these extracted serotype antigens may be used in a simple capillary precipitin method. This method uses unadsorbed, undiluted rabbit antisera. In the capillary precipitin method a 3+ to 4+ reaction was considered significant. The microbactericidal assay and capillary precipitin methods for serotyping group B meningococci show excellent agreement. Group B meningococci were also serotyped by an agar gel double diffusion technique. The latter technique conserves reagents and has the further advantage that it does not show the minor cross-reactions observed in the capillary precipitin method. Thus two simple, reproducible methods for serological typing of group B meningococci have been developed. These methods developed for the serological typing of group B meningococci will aid in epidemiological studies of meningococcal disease. They may also be of value for selecting suitable strains for vaccine production.
Effect of Catecholamines on the Bactericidal Activity of Polymorphonuclear Leukocytes
Authors: Qualliotine Diane , DeChatelet Lawrence R. , McCall Charles E. , Cooper M. Robert
Published: January 2020

Epinephrine, norepinephrine, and dihydroxyphenylalanine at 8 m
m
concentrations prevented iodination of zymosan by intact neutrophils and decarboxylation of
l
-alanine by leukocyte sonic extracts. The same concentration of epinephrine also reduced bactericidal activity of the leukocyte against
Staphylococcus aureus, Enterobacter cloacae
, and
Proteus rettgeri
without decreasing phagocytosis of bacteria. Spectral studies indicated that epinephrine interferes with the myeloperoxidase-mediated reactions by competing for available H
2
O
2
via its enzymatic oxidation to adrenochrome. These findings support a mechanism in which H
2
O
2
plays an important role in the bactericidal activity of the leukocyte.