bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yops ofYersinia enterocoliticaInhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes
Authors: Visser L. G. , Seijmonsbergen E. , Nibbering P. H. , van den Broek P. J. , van Furth R. 
Published: December 2019

ABSTRACTThe virulence plasmid-borne genes encodingYersiniaadhesin A (YadA) and severalYersiniasecreted proteins (Yops) are involved in the inhibition of phagocytosis and killing ofYersinia enterocoliticaby human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis ofY. enterocoliticaby human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+)Y. enterocoliticainhibits O2−production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2−production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochromec. PYV+Y. enterocoliticainhibited O2−production by granulocytes incubated with opsonizedY. enterocoliticaorN-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2−production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2−production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of theyopHgene abrogated the inhibition of phagocytosis of antibody- and complement-opsonizedY. enterocoliticaby human granulocytes but not of the f-MLP-induced O2−production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved.
Role of the Extracellular Signal-Regulated Protein Kinase Cascade in Human Neutrophil Killing ofStaphylococcus aureusandCandida albicansand in Migration
Authors: Hii Charles S. T. , Stacey Kathryn , Moghaddami Nahid , Murray Andrew W. , Ferrante Antonio 
Published: December 2019

ABSTRACTKilling ofStaphylococcus aureusandCandida albicansby neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing ofS. aureusandC. albicanshas not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to killS. aureusandC. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to killS. aureusorC. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.
Human Granulocytic Ehrlichiosis Agent andEhrlichia chaffeensisReside in Different Cytoplasmic Compartments in HL-60 Cells
Authors: Mott Jason , Barnewall Roy E. , Rikihisa Yasuko 
Published: December 2019

ABSTRACTThe human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia,Ehrlichia chaffeensis, in HL-60 cells. Although bothEhrlichiaspp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of bothEhrlichiaspp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not aroundE. chaffeensisinclusions. 3-(2,4-Dinitroanilino)-3′-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized withE. chaffeensisinclusions.E. chaffeensisinclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent andE. chaffeensisinclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; α-adaptin; clathrin heavy chain; or β-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of eitherEhrlichiasp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, whileE. chaffeensisresides in an early endosomal compartment which accumulates the transferrin receptor.
Characterization of Human Bactericidal Antibodies toBordetella pertussis
Authors: Weiss Alison A. , Mobberley Paula S. , Fernandez Rachel C. , Mink ChrisAnna M. 
Published: December 2019

ABSTRACTTheBordetella pertussisBrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure toB. pertussis(infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure toB. pertussisor cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvgmutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS orbvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS ofB. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement.
Chlamydia pneumoniaeInfection in Human Monocytes
Authors: Airenne Sari , Surcel Heljä-Marja , Alakärppä Hannu , Laitinen Kirsi , Paavonen Jorma , Saikku Pekka , Laurila Aino 
Published: December 2019

ABSTRACTChlamydia pneumoniaeinfection has been associated with cardiovascular diseases in seroepidemiological studies and by demonstration of the pathogen in atherosclerotic lesions. It has the capacity to infect several cell types, including monocyte-derived macrophages, which play an essential role in the development of atherosclerosis. However, the persistence ofC. pneumoniaein mononuclear cells is poorly understood. To study the morphology and biological characteristics of the infection, human peripheral blood monocytes were infected withC. pneumoniae. Freshly isolated monocytes resisted the development of infectious progeny, and confocal and transmission electron microscopy showed that the morphology of the inclusions and chlamydial particles was abnormal. Addition of tryptophan or antibodies against gamma interferon did not diminish the inhibition ofC. pneumoniae, suggesting that other factors are involved in the chlamydiostatic activity of the monocytes. Chlamydial mRNA was expressed at least 3 days after infection, however, and a capability for infected monocytes to induce a positive lymphocyte proliferative response was detected for up to 7 days, indicating thatC. pneumoniaeremains metabolically active in the monocytes in vitro. These results are in accordance with the hypothesis thatC. pneumoniaemay participate in the maintenance of local immunological response and inflammation via infected monocytes and thus enhance atherosclerosis.
Interleukin-8 Controls Bacterial Transepithelial Translocation at the Cost of Epithelial Destruction in Experimental Shigellosis
Authors: Sansonetti P. J. , Arondel J. , Huerre M. , Harada A. , Matsushima K. 
Published: December 2019

ABSTRACTIn shigellosis, the network of cellular interactions mediated by a balance of pro- and anti-inflammatory cytokines or chemokines is clearly tipped toward acute destructive inflammation of intestinal tissues by the bacterial invader. This work has addressed the role played by interleukin-8 (IL-8) in a rabbit model of intestinal invasion byShigella flexneri. IL-8, which is largely produced by the epithelial cells themselves, appears to be a major mediator of the recruitment of polymorphonuclear leukocytes (PMNs) to the subepithelial area and transmigration of these cells through the epithelial lining. Neutralization of IL-8 function by monoclonal antibody WS-4 caused a decrease in the amount of PMNs streaming through the lamina propria and the epithelium, thus significantly attenuating the severity of epithelial lesions in areas of bacterial invasion. These findings are in agreement with our previous work (31). In contrast to the PMNs, the bacteria displayed increased transepithelial translocation, as well as overgrowth in the lamina propria and increased passage into the mesenteric blood. By mediating eradication of bacteria at their epithelial entry site, although at the cost of severe epithelial destruction, IL-8 therefore appears to be a key chemokine in the control of bacterial translocation.
Cystic Fibrosis Transmembrane Conductance Regulator-Mediated Corneal Epithelial Cell Ingestion of
Pseudomonas aeruginosa
Is a Key Component in the Pathogenesis of Experimental Murine Keratitis
Authors: Zaidi Tanweer S. , Lyczak Jeffrey , Preston Michael , Pier Gerald B. 
Published: December 2019

ABSTRACT

Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for
Pseudomonas aeruginosa
ingestion into respiratory epithelial cells. In experimental murine keratitis,
P. aeruginosa
enters corneal epithelial cells. We determined the importance of CFTR-mediated uptake of
P. aeruginosa
by corneal cells in experimental eye infections. Entry of noncytotoxic (
exoU
)
P. aeruginosa
into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117. Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to
P. aeruginosa
following corneal cell ingestion. In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (
exoU
+
) or noncytotoxic
P. aeruginosa
resulted in large reductions in bacteria in the eye and markedly lessened eye pathology. Compared with wild-type C57BL/6 mice, heterozygous ΔF508
Cftr
mice infected with
P. aeruginosa
had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology. Homozygous ΔF508
Cftr
mice were nearly completely resistant to
P. aeruginosa
corneal infection. CFTR-mediated internalization of
P. aeruginosa
by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung,
P. aeruginosa
uptake by surface epithelial cells enhances
P. aeruginosa
clearance from this tissue.
Characterization of
Moraxella
(
Branhamella
)
catarrhalis lbpB
,
lbpA
, and Lactoferrin Receptor
orf3
Isogenic Mutants
Authors: Bonnah Robert A. , Wong Henry , Loosmore Sheena M. , Schryvers Anthony B. 
Published: December 2019

ABSTRACT

Pathogenic members of the family
Neisseriaceae
produce specific receptors to acquire iron from their host’s lactoferrin and transferrin. Recently, putative
Moraxella catarrhalis
lactoferrin receptor genes and a third open reading frame (
lbpB
,
lbpA
, and
orf3
) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.
Correlation of Temperature and Toxicity in Murine Studies of Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin 1
Authors: Stiles Bradley G. , Campbell Yvette G. , Castle Robert M. , Grove Sara A. 
Published: December 2019

ABSTRACTThis study describes a quick (<12 h) assay for detecting temperature decreases in BALB/c and C57BL/6 mice injected intraperitoneally (i.p.) with staphylococcal enterotoxin A (SEA), SEB, or SEC3 or toxic shock syndrome toxin 1 and a potentiating dose of lipopolysaccharide (LPS). Toxin-specific antisera effectively neutralized the temperature fluctuations in this model. Orally administered SEA or SEB (50 μg/animal), with or without LPS, did not have an effect on temperature or lethality. Versus wild-type mice, transgenic knockout mice lacking the p55 receptor for tumor necrosis factor (TNF) or gamma interferon were protected against an i.p. challenge of SEA plus LPS. The p75 receptor for TNF and intercellular adhesion molecule 1 have a negligible role in this toxic shock model.
Molecular Characterization of a Flagellar Export Locus of
Helicobacter pylori
Authors: Porwollik Steffen , Noonan Brian , O’Toole Paul W. 
Published: December 2019

ABSTRACT

Motility of
Helicobacter
species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in
Helicobacter pylori
. A 7-kb fragment of the
H. pylori
CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (
ileS
and
murB
), flagellar export genes (
fliI
and
fliQ
), and a homolog to a gene implicated in virulence factor transport in other pathogens (
virB11
). A promoter for this operon, showing similarity to the
Escherichia coli
ς
70
consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of
virB11
,
fliI
,
fliQ
, and
murB
was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of
H. pylori
clinical isolates. Engineered
fliI
and
fliQ
mutant strains were completely aflagellate and nonmotile, whereas a
virB11
mutant still produced flagella. The
fliI
and
fliQ
mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in
H. pylori
26695, was reduced in both the
virB11
mutant and the
fliI
mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).
Survival ofEnterococcus faecalisin Mouse Peritoneal Macrophages
Authors: Gentry-Weeks Claudia R. , Karkhoff-Schweizer RoxAnn , Pikis Andreas , Estay Monica , Keith Jerry M. 
Published: December 2019

ABSTRACTEnterococcus faecaliswas tested for the ability to persist in mouse peritoneal macrophages in two separate studies. In the first study, the intracellular survival of serum-passagedE. faecalis418 and two isogenic mutants [cytolytic strain FA2-2(pAM714) and non-cytolytic strain FA2-2(pAM771)] was compared with that ofEscherichia coliDH5α by infecting BALB/c mice intraperitoneally and then monitoring the survival of the bacteria within lavaged peritoneal macrophages over a 72-h period. AllE. faecalisisolates were serum passaged to enhance the production of cytolysin.E. faecalis418, FA2-2(pAM714), and FA2-2(pAM771) survived at a significantly higher level (P= 0.0001) than didE. coliDH5α at 24, 48, and 72 h. InternalizedE. faecalis418, FA2-2(pAM714), and FA2-2(pAM771) decreased 10-, 55-, and 31-fold, respectively, over the 72-h infection period, while internalizedE. coliDH5α decreased 20,542-fold. The difference in the rate of survival ofE. faecalisstrains andE. coliDH5α was most prominent between 6 and 48 h postinfection (P= 0.0001); however, no significant difference in killing was observed between 48 and 72 h postinfection. In the second study, additionalE. faecalisstrains from clinical sources, including DS16C2, MGH-2, OG1X, and the cytolytic strain FA2-2(pAM714), were compared with the nonpathogenic gram-positive bacterium,Lactococcus lactisK1, for the ability to survive in mouse peritoneal macrophages. In these experiments, theE. faecalisstrains andL. lactisK1 were grown in brain heart infusion (BHI) broth to ensure that there were equal quantities of injected bacteria.E. faecalisFA2-2(pAM714), DS16C2, MGH-2, and OG1X survived significantly better (P< 0.0001) than didL. lactisK1 at each time point.L. lactisK1 was rapidly destroyed by the macrophages, and by 24 h postinfection, viableL. lactiscould not be recovered.E. faecalisFA2-2(pAM714), DS16C2, MGH-2, and OG1X declined at an equivalent rate over the 72-h infection period, and there was no significant difference in survival or rate of decline among the strains.E. faecalisFA2-2(pAM714), MGH-2, DS16C2, and OG1X exhibited an overall decrease of 25-, 55-, 186-, and 129-fold respectively, between 6 and 72 h postinfection. The overall reduction by 1.3 to 2.27 log units is slightly higher than that seen for serum-passagedE. faecalisstrains and may be attributable to the higher level of uptake of serum-passagedE. faecalisthan ofE. faecalisgrown in BHI broth. Electron microscopy of infected macrophages revealed thatE. faecalis418 was present within an intact phagocytic vacuole at 6 h postinfection but that by 24 h the infected macrophages were disorganized, the vacuolar membrane was degraded, and the bacterial cells had entered the cytoplasm. Macrophage destruction occurred by 48 h, and the bacteria were released. In conclusion, the results of these experiments indicate thatE. faecaliscan persist for an extended period in mouse peritoneal macrophages.
Ehrlichia chaffeensis
and
E. sennetsu
, but Not the Human Granulocytic Ehrlichiosis Agent, Colocalize with Transferrin Receptor and Up-Regulate Transferrin Receptor mRNA by Activating Iron-Responsive Protein 1
Authors: Barnewall Roy E. , Ohashi Norio , Rikihisa Yasuko 
Published: December 2019

ABSTRACT

Ehrlichia chaffeensis
and
E. sennetsu
are genetically divergent obligatory intracellular bacteria of human monocytes and macrophages, and the human granulocytic ehrlichiosis (HGE) agent is an obligatory intracellular bacterium of granulocytes. Infection with both
E. chaffeensis
and
E. sennetsu
, but not HGE agent, in the acute monocytic leukemia cell line THP-1 almost completely inhibited by treatment with deferoxamine, a cell-permeable iron chelator. Transferrin receptors (TfRs) accumulated on both
E. chaffeensis
and
E. sennetsu
, but not HGE agent, inclusions in THP-1 cells or the cells of the promyelocytic leukemia cell line HL-60. Reverse transcription-PCR showed an increase in the level of TfR mRNA 6 h postinfection which peaked at 24 h postinfection with both
E. chaffeensis
and
E. sennetsu
infection in THP-1 or HL-60 cells. In contrast, HGE agent in THP-1 or HL-60 cells induced no increase in TfR mRNA levels. Heat treatment of
E. chaffeensis
or the addition of monodansylcadaverine, a transglutaminase inhibitor, 3 h prior to infection inhibited the up-regulation of TfR mRNA. The addition of oxytetracycline 6 h after
E. chaffeensis
infection caused a decrease in TfR mRNA which returned to the basal level by 24 h postinfection. These results indicate that both internalization and continuous proliferation of ehrlichial organisms or the production of ehrlichial proteins are required for the up-regulation of TfR mRNA. Results of electrophoretic mobility shift assays showed that both
E. chaffeensis
and
E. sennetsu
infection increased the binding activity of iron-responsive protein 1 (IRP-1) to the iron-responsive element at 6 h postinfection and remained elevated at 24 h postinfection. However, HGE agent infection had no effect on IRP-1 binding activity. This result suggests that activation of IRP-1 and subsequent stabilization of TfR mRNA comprise the mechanism of TfR mRNA up-regulation by
E. chaffeensis
and
E. sennetsu
infection.
Membrane Topology and Cellular Location of theTreponema pallidumGlycerophosphodiester Phosphodiesterase (GlpQ) Ortholog
Authors: Shevchenko Dmitriy V. , Sellati Timothy J. , Cox David L. , Shevchenko Olga V. , Robinson Esther J. , Radolf Justin D. 
Published: December 2019

ABSTRACTRecent reports that isolatedTreponema pallidumouter membranes contain an ortholog for glycerophosphodiester phosphodiesterase (GlpQ) (D. V. Shevchenko, D. R. Akins, E. J. Robinson, M. Li, O. V. Shevchenko, and J. D. Radolf, Infect. Immun. 65:4179–4189, 1997) and that this protein is a potential opsonic target forT. pallidum(C. E. Stebeck, J. M. Shaffer, T. W. Arroll, S. A. Lukehart, and W. C. Van Voorhis, FEMS Microbiol. Lett. 154:303–310, 1997) prompted a more detailed investigation of its physicochemical properties and cellular location. [14C]palmitate radiolabeling studies of a GlpQ-alkaline phosphatase fusion expressed inEscherichia coliconfirmed the prediction from DNA sequencing that the protein is lipid modified. Studies using Triton X-114 phase partitioning revealed that the protein’s amphiphilicity is due to lipid modification and that a substantial portion of the polypeptide is associated with theT. pallidumpeptidoglycan sacculus. Three different approaches, i.e., (i) proteinase K treatment of intact treponemes, (ii) indirect immunofluorescence analysis of treponemes encapsulated in agarose beads, and (iii) opsonophagocytosis of treponemes incubated with antiserum against recombinant GlpQ by rabbit peritoneal macrophages, confirmed that GlpQ is entirely subsurface inT. pallidum. Moreover, rabbits hyperimmunized with GlpQ were not protected against intradermal challenge with virulent treponemes. Circular dichroism spectroscopy confirmed that the recombinant form of the polypeptide lacked discernible evidence of denaturation. Finally, GlpQ was not radiolabeled whenT. pallidumouter membranes were incubated with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarene, a photoactivatable, lipophilic probe which promiscuously labels both proteins and lipids within phospholipid bilayers. Taken as a whole, these studies indicate that theT. pallidumGlpQ ortholog is a periplasmic protein associated predominantly with the spirochete’s peptidoglycan-cytoplasmic membrane complex.