bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Flagella in Pathogenesis ofPseudomonas aeruginosaPulmonary Infection
Authors: Feldman Matthew , Bryan Ruth , Rajan Sujatha , Scheffler Lee , Brunnert Steven , Tang Hope , Prince Alice
Published: December 2019

Pseudomonas aeruginosastrains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens,P. aeruginosacells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence offliCmutants in a neonatal mouse model of pneumonia. In the absence offliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to apilAmutant PAK/NP (P< 0.0001). ThefliCmutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that thefliCmutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung.125I-labeled flagellin bound to the glycolipids GM1 and GD1aand to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type orfliCmutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla+organisms but notfliCmutants were efficiently taken up by murine macrophages.P. aeruginosaflagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.
Phage Antibodies Obtained by Competitive Selection on Complement-ResistantMoraxella(Branhamella)catarrhalisRecognize the High-Molecular-Weight Outer Membrane Protein
Authors: Boel Edwin , Bootsma Hester , de Kruif John , Jansze Margriet , Klingman Karin L. , van Dijk Hans , Logtenberg Ton
Published: December 2019

We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcusMoraxella(Branhamella)catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) ofM. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitiveM. catarrhalisstrains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.
Processing ofMycobacterium tuberculosisBacilli by Human Monocytes for CD4+αβ and γδ T Cells: Role of Particulate Antigen
Authors: Balaji Kithiganahalli N. , Boom W. Henry
Published: December 2019

Mycobacterium tuberculosisreadily activates both CD4+and Vδ2+γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented byM. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing ofM. tuberculosisantigens to human CD4 and γδ T cells by monocytes. Initial uptake ofM. tuberculosisbacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2+γδ T cells. For γδ T cells, recognition ofM. tuberculosis-infected monocytes was dependent on Vδ2+T-cell-receptor expression. Recognition ofM. tuberculosisantigens by CD4+T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing ofM. tuberculosisbacilli for Vδ2+γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing ofM. tuberculosisbacilli for CD4+T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing ofM. tuberculosisbacilli for CD4+and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4+T cells. Chloroquine and ammonium chloride insensitivity of processing ofM. tuberculosisbacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4+and γδ T cells.
Filament Tip-Associated Antigens Involved in Adherence to and Invasion of Murine Pulmonary Epithelial Cells In Vivo and HeLa Cells In Vitro by
Nocardia asteroides
Authors: Beaman Blaine L. , Beaman LoVelle
Published: December 2019

The interactions of
Nocardia asteroides
GUH-2 with pulmonary epithelial cells of C57BL/6 mice and with HeLa cells were studied. Electron microscopy demonstrated that only the tips of log-phase cells penetrated pulmonary epithelial cells following intranasal administration, and nocardiae were recovered from the brain. Coccobacillary cells neither invaded nor disseminated. Serum from immunized mice (IMS) decreased attachment to and penetration of pulmonary epithelial cell surfaces by log-phase GUH-2 and inhibited spread to the brain. IMS was adsorbed against stationary-phase cells. Western immunoblots suggested that this adsorbed IMS was reactive primarily with 43- and 62-kDa proteins. Immunofluorescence showed that adsorbed IMS preferentially labeled the tips of log-phase GUH-2 cells. Since this IMS was reactive to culture filtrate antigens, several of these proteins were cut from gels, and mice were immunized. Sera against 62-, 55-, 43-, 36-, 31-, and 25-kDa antigens were obtained. The antisera against the 43- and 36-kDa proteins labeled the filament tips of GUH-2 cells. Only the antiserum against the 43-kDa antigen increased pulmonary clearance, inhibited apical attachment to and penetration of pulmonary epithelial cells, and prevented spread to the brain. An in vitro model with HeLa cells demonstrated that the tips of log-phase cells of GUH-2 adhered to and penetrated the surface of HeLa cells. Invasion assays with amikacin treatment demonstrated that nocardiae were internalized. Adsorbed IMS blocked attachment to and invasion of these cells. These data suggested that a filament tip-associated 43-kDa protein was involved in attachment to and invasion of pulmonary epithelial cells and HeLa cells by
N. asteroides
GUH-2.
Anthrax Toxin as a Molecular Tool for Stimulation of Cytotoxic T Lymphocytes: Disulfide-Linked Epitopes, Multiple Injections, and Role of CD4+Cells
Authors: Ballard Jimmy D. , Collier R. John , Starnbach Michael N.
Published: December 2019

We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4+helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.
Characterization of Outer Membrane Protein OmpU of
Vibrio cholerae
O1
Authors: Nakasone Noboru , Iwanaga Masaaki
Published: December 2019

The outer membrane protein OmpU of
Vibrio cholerae
O1 strain 86B3 was characterized with reference to colonization of the intestine by the organism. The purified OmpU exhibited a pI of 3.6. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated to 38, 32, and 110 kDa when the sample was heated at 100°C for 2 min, 50°C for 15 min, and room temperature for 30 min, respectively. The purified OmpU was not hemagglutinative. Anti-OmpU serum did not agglutinate strain 86B3 or other
V. cholerae
organisms. OmpU adhered to the brush border of the rabbit small intestine; adhesion of the organisms to the intestine treated in advance with OmpU was not inhibited. Treating the organisms in advance with anti-OmpU Fab did not inhibit adhesion to the intestine. These results obtained in vitro suggest that OmpU is not involved in the adhesion of
V. cholerae
to the intestinal epithelium.
Structure and Specific Activity of Macrophage-Stimulating Lipopeptides fromMycoplasma hyorhinis
Authors: Mühlradt Peter F. , Kiess Michael , Meyer Holger , Süssmuth Roderich , Jung Günther
Published: December 2019

Mycoplasmas are potent macrophage stimulators. We describe the isolation of macrophage-stimulatory lipopeptidesS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTDNNSSQSQQPGSGTTNT andS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTN derived from theMycoplasma hyorhinisvariable lipoproteins VlpA and VlpC, respectively. These lipopeptides were characterized by amino acid sequence and composition analysis and by mass spectrometry. The lipopeptidesS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-GQTNT andS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-SKKKK and the N-palmitoylated derivative of the latter were synthesized, and their macrophage-stimulatory activities were compared in a nitric oxide release assay with peritoneal macrophages from C3H/HeJ mice. The lipopeptides with the free amino terminus showed half-maximal activity at 3 pM regardless of their amino acid sequence; i.e., they were as active as the previously isolatedM. fermentans-derived lipopeptide MALP-2. The macrophage-stimulatory activity of the additionally N-palmitoylated lipopeptide or of the murein lipoprotein fromEscherichia coli, however, was lower by orders of magnitude. It is concluded that the lack of N-acyl groups in mycoplasmal lipoproteins explains their exceptionally high in vitro macrophage-stimulatory capacity. Certain features that lipopolysaccharide endotoxin and mycoplasmal lipopeptides have in common are discussed. Lipoproteins and lipopeptides are likely to be the main causative agents of inflammatory reactions to mycoplasmas. This may be relevant in the context of mycoplasmas as arthritogenic pathogens and their association with AIDS.
Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain fromPichia pastorisas a Recombinant Vaccine Candidate
Authors: Byrne Michael P. , Smith Theresa J. , Montgomery Vicki A. , Smith Leonard A.
Published: December 2019

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed inPichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 μg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 μg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of ≥0.5 μg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of ≥1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of ≤100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.
Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro
Authors: Winram Scott B. , Jonas Mechthild , Chi Emil , Rubens Craig E.
Published: December 2019

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol.
87:
779–784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics
61:
171–177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.
Biological and Biochemical Characteristics of Cytoadhesion of
Plasmodium falciparum
-Infected Erythrocytes to Chondroitin-4-Sulfate
Authors: Pouvelle Bruno , Fusaï Thierry , Lépolard Catherine , Gysin Jürg
Published: December 2019

The cytoadhesion of
Plasmodium falciparum
laboratory strains and clones to
Saimiri
brain microvascular endothelial cells (SBEC 17), with chondroitin-4-sulfate (CSA) as the only adhesion receptor, was tested. Only one strain had significant cytoadhesion. However, CSA-specific infected erythrocytes (IRBCs) were detected in all strains after selection of a CSA-specific subpopulation by culturing the few adherent IRBCs. This demonstrates the lack of sensitivity of cytoadhesion microassays for detecting small quantities of CSA-specific IRBCs in cultures or field isolates. Cytoadhesion to CSA is maximal at 24 h of the cycle and decreases with the onset of schizogony, reaching a minimum just before reinvasion. This fluctuation must be taken into account in comparisons of the cytoadhesion of different strains or isolates. The minimum size of CSA for active inhibition was 4 kDa, and a mass of 9 kDa was required for inhibition similar to that obtained with the 50-kDa CSA. In contrast to cytoadhesion to CSA, which is pH independent or maximal at physiological pH (depending on the target endothelial cells), adhesion to CD36 and intercellular adhesion molecule 1 was pH dependent, requiring acidic conditions to be maximal in all cases. Cytoadhesion to CSA may trigger the occlusion of microvessels and cause the acidosis necessary for the other receptors to be fully efficient. If this key role in the mechanisms of sequestration were to be confirmed in vivo, prevalence studies of the CSA cytoadhesion phenotype would have to be reevaluated, because simple cytoadhesion assays do not detect CSA-specific parasites present in very low numbers, and these parasites might then be undetected in the peripheral blood but present in organs in which sequestration occurs, such as the placenta (M. Fried and P. E. Duffy, Science 272:1502–1504, 1996).
The Cell Cycle-Specific Growth-Inhibitory Factor Produced byActinobacillus actinomycetemcomitansIs a Cytolethal Distending Toxin
Authors: Sugai Motoyuki , Kawamoto Toru , Pérès Sylvie Y. , Ueno Yoko , Komatsuzawa Hitoshi , Fujiwara Tamaki , Kurihara Hidemi , Suginaka Hidekazu , Oswald Eric
Published: December 2019

Actinobacillus actinomycetemcomitanshas been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified inA. actinomycetemcomitansY4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced byHaemophilus ducreyi. The CDT fromA. actinomycetemcomitanswas produced inEscherichia coliand was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by thecdtlocus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions ofA. actinomycetemcomitansY4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions ofA. actinomycetemcomitansY4. This CDT is a new putative virulence factor ofA. actinomycetemcomitansand may play a role in the pathogenesis of periodontal diseases.
Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against LethalTrypanosoma cruziInfection
Authors: Wizel Benjamin , Garg Nisha , Tarleton Rick L.
Published: December 2019

DNA vaccination was evaluated with the experimental murine model ofTrypanosoma cruziinfection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruziantibody and major histocompatibility complex (MHC) class I-restricted CD8+cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization ofH-2bandH-2dmice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge withT. cruziwere assessed. InH-2bmice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protectiveH-2Kb-restricted TSA-1515–522epitope. InH-2dmice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysedT. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8+-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged withT. cruzi, 14 of 22 (64%)H-2band 16 of 18 (89%)H-2dmice survived the infection. The ability to induce significant murine anti-T. cruziprotective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzivaccines which may ultimately be used for the prevention or treatment of Chagas’ disease.
Altered Immune Responses in Mice with ConcomitantSchistosoma mansoniandPlasmodium chabaudiInfections
Authors: Helmby Helena , Kullberg Marika , Troye-Blomberg Marita
Published: December 2019

Mixed parasitic infections are common in many parts of the world. However, little is known about how concurrent infections affect the immunity to and/or pathogenesis of each other. Protection and elimination of blood-stagePlasmodium chabaudi chabaudiAS in resistant mice are characterized by a sequential activation of CD4+Th1 and Th2 cells. The patent egg-laying stage of the murine model ofSchistosoma mansoniis associated with a strong Th2 response to bothSchistosomaand unrelated antigens. In this study, we investigated how infection of mice withS. mansoniwould affect the immune response to and pathogenesis of aP. chabaudiinfection. C57BL/6 mice infected withS. mansonifor 8 weeks were infected with blood-stageP. chabaudi. Malaria parasitemias were significantly higher in these mice than in mice infected withP. chabaudionly. In doubly infected mice, both spleen cell proliferative and Th2 responses toS. mansonisoluble egg antigen (SEA) or anti-CD3 were suppressed up to 1 month after the malaria infection. Findings for SEA-specific immunoglobulin M (IgM) and IgG serum antibody levels were similar. No significant effects were seen onP. chabaudi-induced gamma interferon responses. However, tumor necrosis factor alpha (TNF-α) production was significantly lower in double-infected mice. Thus, a defect in TNF-α production might contribute to the increased malaria parasitemias seen inS. mansoni-P. chabaudi-infected mice. Taken together, our data show that schistosoma and malaria infections profoundly affect each other, findings which might have implications for the development of vaccines.
Resident Enteric Bacteria Are Necessary for Development of Spontaneous Colitis and Immune System Activation in Interleukin-10-Deficient Mice
Authors: Sellon Rance K. , Tonkonogy Susan , Schultz Michael , Dieleman Levinus A. , Grenther Wetonia , Balish Ed , Rennick Donna M. , Sartor R. Balfour
Published: December 2019

Mice with targeted deletion of the gene for interleukin-10 (IL-10) spontaneously develop enterocolitis when maintained in conventional conditions but develop only colitis when kept in specific-pathogen-free (SPF) environments. This study tested the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice. IL-10-deficient mice were maintained in either SPF conditions or germfree conditions or were populated with bacteria known to cause colitis in other rodent models. IL-10-deficient mice kept in SPF conditions developed colitis in all segments of the colon (cecum and proximal and distal colon). These mice exhibited immune system activation as evidenced by increased expression of CD44 on CD4+T cells; increased mesenteric lymph node cell numbers; and increased production of immunoglobulin A (IgA), IgG1, and IL-12 p40 from colon fragment cultures. Mice populated with bacterial strains, includingBacteroides vulgatus, known to induce colitis in other rodent models had minimal colitis. Germfree IL-10-deficient mice had no evidence of colitis or immune system activation. We conclude therefore that resident enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice.
Regulation of Protease Expression in
Porphyromonas gingivalis
Authors: Tokuda Masayuki , Chen Wen , Karunakaran Thonthi , Kuramitsu Howard K.
Published: December 2019

Although the strong protease activity of
Porphyromonas gingivalis
appears to be an important virulence property of these organisms, little information is currently available regarding the regulation of expression of the multiple protease genes. Utilizing the
lacZ
reporter gene strategy, the environmental factors which regulate the expression of the Arg-gingipain gene
rgpA
and the
prtT
protease gene were investigated. These two genes are reciprocally regulated since factors which retarded growth (iron depletion and nutrient limitation) appeared to upregulate
rgpA
expression while down-regulating
prtT
expression. However, inactivation of the major
rgpA
gene resulted in increased transcription of the
prtT
and
tpr
protease genes while decreasing expression of the Lys-gingipain
kgp
gene as detected by Northern blot analysis. By contrast, inactivation of the
prtT
gene did not significantly affect
kgp
expression but moderately decreased
rgpA
mRNA levels. These results indicate that the protease genes of
P. gingivalis
are not coordinately regulated and suggest that some of these enzymes play specific roles in the physiology and/or virulence of these organisms.
Staphylococcus aureus
Agr and Sar Global Regulators Influence Internalization and Induction of Apoptosis
Authors: Wesson Carla A. , Liou Linda E. , Todd Kristine M. , Bohach Gregory A. , Trumble William R. , Bayles Kenneth W.
Published: December 2019

Staphylococcus aureus
was recently shown to be internalized by and to induce apoptosis in a bovine mammary epithelial cell line, suggesting that these processes could be involved in staphylococcal pathogenesis or persistence. To examine the role of virulence factor regulators during internalization, mutant
agr
and
sar
strains of
S. aureus
were analyzed for their abilities to enter and induce apoptosis in epithelial cells. Like a previously characterized bovine mastitis isolate, the standard laboratory strain, RN6390 (wild type), entered the epithelial cells and subsequently induced apoptosis. In contrast, the mutant strains RN6911 (
agr
), ALC136 (
sar
), and ALC135 (
agr sar
) were internalized by the cultured cells at levels reproducibly greater than that for RN6390 but failed to induce apoptosis. The internalization of
S. aureus
was affected by growth phase, suggesting a role for
agr
-regulated surface proteins in this process. Furthermore, the ability to induce apoptosis required metabolically active intracellular bacteria. These data indicate that the ability of
S. aureus
to enter mammalian cells and induce apoptosis is dependent on factors regulated by Agr and Sar. Since transcriptional control by these global regulators is mediated by quorum-sensing and environmental factors, staphylococci may have the potential to induce several alternative effects on cells from an intracellular environment. A model for the function of the
agr
locus in the context of internalization, intracellular persistence, and dissemination is proposed.
The p29 and p35 Immunodominant Antigens ofNeospora caninumTachyzoites Are Homologous to the Family of Surface Antigens ofToxoplasma gondii
Authors: Howe Daniel K. , Crawford Amy C. , Lindsay David , Sibley L. David
Published: December 2019

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondiiand has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) fromN. caninumtachyzoites that are the predominant antigens recognized by antisera fromNeospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverseN. caninumisolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to theT. gondiisurface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins ofT. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.
Fusion of
Chlamydia trachomatis
-Containing Inclusions Is Inhibited at Low Temperatures and Requires Bacterial Protein Synthesis
Authors: Van Ooij Christiaan , Homola Ellen , Kincaid Eleanor , Engel Joanne
Published: December 2019

The human pathogen
Chlamydia trachomatis
is an obligate intracellular bacterium with a unique developmental cycle. Within the host cell cytoplasm, it resides within a membrane-bound compartment, the inclusion. A distinguishing characteristic of the
C. trachomatis
life cycle is the fusion of the chlamydia-containing inclusions with each other in the host cell cytoplasm. We report that fusion of inclusions does not occur at 32°C in multiple mammalian cell lines and with three different serovars of
C. trachomatis
. The inhibition of fusion was inclusion specific; the fusion with sphingolipid-containing secretory vesicles and the interaction with early endosomes were unaffected by incubation at 32°C. The inhibition of fusion of the inclusions was not primarily the result of delayed maturation of the inclusion, as infectious progeny was produced in host cells incubated at 32°C, and the unfused inclusions remained competent to fuse up to 48 h postinfection. The ability to reverse the inhibition of fusion by shifting the infected cells from 32 to 37°C allowed the measurement of the rate and the time of fusion of the inclusions after entry of the bacteria. Most significantly, we demonstrate that fusion of inclusions with each other requires bacterial protein synthesis and that the required bacterial protein(s) is present, but inactive or not secreted, at 32°C.
A Streptococcal Adhesion System for Salivary Pellicle and Platelets
Authors: Gong Ke , Ouyang T. , Herzberg M. C.
Published: December 2019

A
Streptococcus sanguis
133-79 adhesin identified by the monoclonal antibody 1.1 (MAb 1.1) binds both saliva-coated hydroxylapatite (sHA) and platelets. The complementary binding site(s) for the adhesin was identified by the anti-idiotypical MAb 2.1. To learn if this adhesion system, marked by the antiadhesin MAb 1.1 and anti-binding site MAb 2.1, is commonly used by strains within the sanguis group and other viridans group streptococci, 42 strains from seven species were tested. Strains that bind to both sHA and platelets use the same adhesin and binding site epitopes. Strains that do not adhere to platelets rely on other adhesin specificities to bind to sHA.
Gamma Interferon Production by Cytotoxic T Lymphocytes Is Required for Resolution ofChlamydia trachomatisInfection
Authors: Lampe Mary F. , Wilson Christopher B. , Bevan Michael J. , Starnbach Michael N.
Published: December 2019

In this study, we used mice in which the gene for gamma interferon (IFN-γ) has been disrupted (IFN-γ−/−mice) to study the role of this cytokine in the resolution ofChlamydia trachomatisinfection. We show that IFN-γ−/−mice are impaired in the ability to clear infection withC. trachomatiscompared to IFN-γ+/+control mice. Activated CD8+cytotoxic T lymphocytes (CTL) secrete IFN-γ in response to intracellular infection, and we have shown previously that aChlamydia-specific CTL line can reduceC. trachomatisinfection when adoptively transferred into infected mice. In the present study, we found that when these IFN-γ+/+CTL lines are transferred intoChlamydia-infected IFN-γ−/−mice, the transferred CTL cannot overcome the immune defect seen in the IFN-γ−/−mice. We also show thatChlamydia-specific CTL can be cultured from IFN-γ-deficient mice infected withC. trachomatis; however, the adoptive transfer of IFN-γ−/−CTL into infected IFN-γ+/+mice does not reduce the level of infection. These results suggest that IFN-γ production by CTL is not sufficient to overcome the defect that IFN-γ−/−mice have in the resolution ofChlamydiainfection, yet IFN-γ production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-γ+/+mice.
Deletion of the Major Proteolytic Site of the
Helicobacter pylori
Cytotoxin Does Not Influence Toxin Activity but Favors Assembly of the Toxin into Hexameric Structures
Authors: Burroni Daniela , Lupetti Pietro , Pagliaccia Cristina , Reyrat Jean Marc , Dallai Romano , Rappuoli Rino , Telford John L.
Published: December 2019

The
Helicobacter pylori
cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.
A Protein (M9) Associated with Monoclonal Antibody-Mediated Agglutination ofMycoplasma gallisepticumIs a Member of the pMGA Family
Authors: Liu Li , Payne D. Michael , van Santen Vicky L. , Dybvig Kevin , Panangala Victor S.
Published: December 2019

A 62-kDa cell surface antigen (M9) ofMycoplasma gallisepticumPG31 that mediates antibody-induced agglutination of the organism was purified and subjected to N-terminal amino-acid sequencing. A 999-bp region of the cDNA encoding the M9 protein was generated by reverse transcription-PCR, and its nucleotide sequence was determined. PCR primers based on this sequence were used to screen a genomic DNA library of PG31. A full-length M9 protein-encoding gene was isolated and sequenced, revealing 96% nucleotide identity with thepMGA1.1gene ofM. gallisepticumS6. Sequence analyses of theM9gene and flanking open reading frames that encode other pMGA family members suggest that a tandemly repeated GAA sequence may influencepMGAgene expression.
Brucella abortus
Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes
Authors: Pizarro-Cerdá Javier , Méresse Stéphane , Parton Robert G. , van der Goot Gisou , Sola-Landa Alberto , Lopez-Goñi Ignacio , Moreno Edgardo , Gorvel Jean-Pierre
Published: December 2019

Brucella abortus
is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent
B. abortus
strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from
Aeromonas hydrophila
causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic
B. abortus
exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.
Relationship of Structure and Biological Activity of Monosaccharide Lipid A Analogues to Induction of Nitric Oxide Production by Murine Macrophage RAW264.7 Cells
Authors: Funatogawa Keiji , Matsuura Motohiro , Nakano Masayasu , Kiso Makoto , Hasegawa Akira
Published: December 2019

Lipid A is the active center of bacterial lipopolysaccharide (LPS), which exhibits diverse biological activities via the production of various mediators. We investigated the production of nitric oxide (NO), one of the mediators, by a murine macrophage cell line, RAW264.7, upon stimulation with a series of monosaccharide lipid A analogues to elucidate the relationship of structure and activity in NO production. The production of other representative mediators, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was also investigated to compare the structural requirements for the production of these cytokines with those for the production of NO. Structure-activity relationships in NO production correlated well with those in the production of TNF-α and IL-6. Among the lipid A analogues possessing different numbers of acyl groups on a 4-O-phosphono-d-glucosamine backbone, compounds like GLA-60 that possess three tetradecanoyl (C14) groups exhibited stronger activities in the production of the mediators than compounds possessing four or two C14groups. Time course study of the production of these mediators showed that production of NO started and peaked later than those of TNF-α and IL-6. Neither neutralization of TNF-α activity by antibody nor suppression of TNF-α production by pentoxifylline showed a significant suppressive effect on production of NO and IL-6 upon stimulation with LPS or lipid A analogues. Neutralization of IL-6 activity by antibody showed no significant suppressive effect on production of NO and TNF-α. A monosaccharide lipid A analogue (GLA-58) which exhibited no detectable agonistic activity showed a suppressive effect on the production of all three mediators upon stimulation with LPS or lipid A analogues. These results indicate that signals for NO production by LPS agonists in murine macrophages are transduced in good correlation with those for production of TNF-α and IL-6, although they are not transduced via production of those cytokines.
Characterization of Membrane-Associated
Clostridium perfringens
Enterotoxin following Pronase Treatment
Authors: Wieckowski Eva U. , Kokai-Kun John F. , McClane Bruce A.
Published: December 2019

After binding,
Clostridium perfringens
enterotoxin (CPE) initially localizes in a small (∼90-kDa) complex in plasma membranes. This event is followed by formation of a second membrane complex, referred to as large (160-kDa) complex. Contrary to a previous hypothesis proposing that CPE inserts into intestinal brush border membranes (BBMs) when this toxin is localized in the small complex, this study shows that BBMs do not offer CPE localized in the small complex protection from pronase. However, our experiments indicate that BBMs do substantially protect CPE from pronase when this toxin is localized in large complex. Since the onset of CPE-induced permeability alterations closely coincides with large-complex formation, these new results suggest that CPE-induced alterations in permeability may result from pore formation due to the partial membrane insertion of CPE when this toxin is present in large complex.
Detection of Anionic Antimicrobial Peptides in Ovine Bronchoalveolar Lavage Fluid and Respiratory Epithelium
Authors: Brogden Kim A. , Ackermann Mark , Huttner Kenneth M.
Published: December 2019

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 ± 0.33 mM AP (mean ± standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 μg/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.
Actinobacillus actinomycetemcomitansToxin Induces Both Cell Cycle Arrest in the G2/M Phase and Apoptosis
Authors: Ohguchi Masahiro , Ishisaki Akira , Okahashi Nobuo , Koide Masanori , Koseki Takeyoshi , Yamato Kenji , Noguchi Toshihide , Nishihara Tatsuji
Published: December 2019

We found that the culture supernatant of the periodontopathic bacteriumActinobacillus actinomycetemcomitanshad a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant ofA. actinomycetemcomitansY4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin fromA. actinomycetemcomitansinduces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin fromA. actinomycetemcomitanscould mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue.
Tumor Necrosis Factor Alpha Enhances Antifungal Activities of Polymorphonuclear and Mononuclear Phagocytes againstAspergillus fumigatus
Authors: Roilides Emmanuel , Dimitriadou-Georgiadou Anastasia , Sein Tin , Kadiltsoglou Isaac , Walsh Thomas J.
Published: December 2019

Invasive aspergillosis is a serious complication in immunocompromised patients. The effects of recombinant human tumor necrosis factor alpha (TNF-α) on antifungal activities of human neutrophils (polymorphonuclear leukocytes [PMNs]), human monocytes (MNCs), and rabbit pulmonary alveolar macrophages (PAMs) againstAspergillus fumigatuswere studied. The percentage of PMN-induced hyphal damage was increased after 30 min of incubation of PMNs with 0.1 ng of TNF-α per ml at 37°C (P= 0.043). At 0.1 to 10 ng/ml, TNF-α also increased superoxide anion (O2−) produced by PMNs in response to phorbol myristate acetate,N-formylmethionyl leucyl phenylalanine, and unopsonized hyphae (P< 0.01) but did not exert any effect on PMN phagocytosis of conidia in the presence of serum. By comparison, TNF-α induced only a slight increase in O2−production by MNCs in response to phorbol myristate acetate (P= 0.05) and no concomitant increase in the percentage of MNC-induced hyphal damage. Incubation of MNCs with TNF-α at 0.001 to 10 ng/ml for 2 days had no effect on phagocytosis or conidiocidal activity. By contrast, incubation of PAMs with TNF-α at 0.1 to 10 ng/ml for 2 days increased phagocytosis of conidia (P= 0.03). Thus, TNF-α augments the capacity of PMNs to damageAspergillushyphae, possibly through enhanced oxidative mechanisms, and increases PAM phagocytic activity against conidia. As such, TNF-α may have an important role in host defense against aspergillosis, and neutralization of its activity may be complicated by increased susceptibility to aspergillosis.
Correlation of Cecal Microflora of HLA-B27 Transgenic Rats with Inflammatory Bowel Disease
Authors: Onderdonk Andrew B. , Richardson James A. , Hammer Robert E. , Taurog Joel D.
Published: December 2019

Transgenic rats with a high level of expression of the human major histocompatibility complex class I molecule HLA-B27 develop chronic inflammatory bowel disease (IBD) and arthritis. Assessment of the cecal microflora showed a rise in numbers ofEscherichia coliandEnterococcusspp., corresponding to the presence and severity of IBD in these rats.
Lactobacilli and Streptococci Induce Interleukin-12 (IL-12), IL-18, and Gamma Interferon Production in Human Peripheral Blood Mononuclear Cells
Authors: Miettinen Minja , Matikainen Sampsa , Vuopio-Varkila Jaana , Pirhonen Jaana , Varkila Kari , Kurimoto Masashi , Julkunen Ilkka
Published: December 2019

Human peripheral blood mononuclear cells (PBMC) were stimulated with three nonpathogenicLactobacillusstrains and with one pathogenicStreptococcus pyogenesstrain, and cytokine gene expression and protein production were analyzed. All bacteria strongly induced interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha mRNA expression and protein production.S. pyogeneswas the most potent inducer of secretion of IL-12 and gamma interferon (IFN-γ), and two of threeLactobacillusstrains induced IL-12 and IFN-γ production. All strains induced IL-18 protein production. IL-10 and IL-4 production was induced weakly and not at all, respectively. Our data show that nonpathogenic lactobacilli and pathogenic streptococci can induce Th1 type cytokines IL-12, IL-18, and IFN-γ in human PBMC.