bluuebunny/crossref_metadata_2025 huggingface dataset:
Chlamydia pneumoniaeInfection in Human Monocytes
Authors: Airenne Sari , Surcel Heljä-Marja , Alakärppä Hannu , Laitinen Kirsi , Paavonen Jorma , Saikku Pekka , Laurila Aino
Published: December 2019

Chlamydia 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

In 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

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

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

This 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

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

Enterococcus 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

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

Recent 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.
Transforming Growth Factor β-Induced Failure of Resistance to Infection with Blood-StagePlasmodium chabaudiin Mice
Authors: Tsutsui Naohisa , Kamiyama Tsuneo
Published: December 2019

The role of transforming growth factor β (TGF-β) in infection withPlasmodium chabaudiwas investigated with resistant and susceptible mouse models. C57BL/10 mice produced gamma interferon (IFN-γ) and nitric oxide (NO) shortly after infection and cleared the parasite spontaneously. In contrast, BALB/c mice showed a transient enhancement of TGF-β production, followed by a relative lack of IFN-γ and NO production, and succumbed to the infection. However, there was no correlation between levels of serum TGF-β and splenic TGF-β mRNA in both mouse strains before and after infection. Administration of recombinant TGF-β (rTGF-β) rendered resistant mice susceptible because of suppression of subsequent production of IFN-γ and NO. Administration of anti-TGF-β antibody to the infected BALB/c mice resulted in remarkable increases in serum IFN-γ and NO, and the mice resisted the infection. Splenic CD4+T and CD11b+cells of C57BL/10 mice were significantly activated after infection, but this was completely abrogated by administration of rTGF-β. These results suggested that, in theP. chabaudi-susceptible but not resistant mice, production of TGF-β was promoted, and subsequent failure of IFN-γ- and NO-dependent resistance to the parasite was induced. This study is the first to indicate that TGF-β production was the key event in failure of resistance to mouse malaria.
Activation of Neutrophil Collagenase in Periodontitis
Authors: Romanelli Raquel , Mancini Sabrina , Laschinger Carol , Overall Christopher M. , Sodek Jaro , McCulloch Christopher A. G.
Published: December 2019

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (
P
< 0.001)-higher levels of active collagenase in periodontitis (
n
= 12) samples compared to gingivitis (
n
= 17) samples, which exhibited low levels of activity, while controls (
n
= 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (
r
2
= 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.
Genetic and Physiologic Characterization of Ferric/Cupric Reductase Constitutive Mutants ofCryptococcus neoformans
Authors: Nyhus Karin J. , Jacobson Eric S.
Published: December 2019

Cryptococcus neoformansis a pathogenic yeast that causes meningitis in immunocompromised patients. Because iron acquisition is critical for growth of a pathogen in a host, we studied the regulation of the ferric reductase and ferrous uptake system of this organism. We isolated 18 mutants, representing four independent loci, with dysregulated ferric reductase. The mutant strains had >10-fold higher than wild-type WT reductase activity in the presence of iron. Two of the strains also had >7-fold higher than WT iron uptake in the presence of iron but were not markedly iron sensitive. Both were sensitive to the oxidative stresses associated with superoxide and hydrogen peroxide. One strain exhibited only 23% of the WT level of iron uptake in the absence of iron and grew poorly without iron supplementation of the medium, phenotypes consistent with an iron transport deficiency; it was sensitive to superoxide but not to hydrogen peroxide. The fourth strain had high reductase activity but normal iron uptake; it was not very sensitive to oxidative stress. We also demonstrated that the ferric reductase was regulated by copper and could act as a cupric reductase. Sensitivity to oxidants may be related to iron acquisition by a variety of mechanisms and may model the interaction of the yeast with the immune system.
SFS, a Novel Fibronectin-Binding Protein fromStreptococcus equi, Inhibits the Binding between Fibronectin and Collagen
Authors: Lindmark Hans , Guss Bengt
Published: December 2019

The obligate parasitic bacteriumStreptococcus equisubsp.equiis the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned fromS. equisubsp.equiand characterized a gene, calledsfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. Thesfsgene was found to be present in all 50 isolates ofS. equisubsp.equitested and in 41 of 48S. equisubsp.zooepidemicusisolates tested. Thesfsgene is down-regulated during growth in vitro compared tofnz, a previously characterized gene encoding an Fn-binding protein fromS. equisubsp.zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the OklahomaS. pyogenesdatabase, we found that this motif is also present in a potential cell surface protein fromS. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way.
Molecular Interactions ofPorphyromonas gingivalisFimbriae with Host Proteins: Kinetic Analyses Based on Surface Plasmon Resonance
Authors: Amano Atsuo , Nakamura Takayuki , Kimura Shigenobu , Morisaki Ichijiro , Nakagawa Ichiro , Kawabata Shigetada , Hamada Shigeyuki
Published: December 2019

Fimbriae ofPorphyromonas gingivalisare thought to play an important role in the colonization and invasion of periodontal tissues. In this study, we analyzed the interactions ofP. gingivalisfimbriae with human hemoglobin, fibrinogen, and salivary components (i.e., proline-rich protein [PRP], proline-rich glycoprotein [PRG], and statherin) based on surface plasmon resonance (SPR) spectroscopy with a biomolecular interaction analyzing system (BIAcore). The real-time observation showed that the fimbriae interacted more quickly with hemoglobin and PRG than with other proteins and more intensely with fibrinogen. The significant association constant (ka) values obtained by BIAcore demonstrated that the interactions between fimbriae and these host proteins are specific. These estimatedKavalues were not too different; however, theKavalues for hemoglobin (2.43 × 106) and fibrinogen (2.16 × 106) were statistically greater than those for the salivary proteins (1.48 × 106to 1.63 × 106). TheKavalue of anti-fimbriae immunoglobulin G for fimbriae was estimated to be 1.22 × 107, which was 6.55-fold higher than the meanKavalue of the host proteins. Peptide PRP-C, a potent inhibitor of PRP-fimbriae interaction, dramatically inhibited fimbrial association to PRP and PRG and was also inhibitory against other host proteins by BIAcore. The binding of fimbriae to these proteins was also evaluated by other methods with hydroxyapatite beads or polystyrene microtiter plates. The estimated binding abilities differed considerably, depending on the assay method that was used. It was noted that the binding capacity of PRP was strongly diminished by immobilization on a polystyrene surface. Taken together, these findings suggest thatP. gingivalisfimbriae possess a strong ability to interact with the host proteins which promote bacterial adherence to the oral cavity and that SPR spectroscopy is a useful method for analyzing specific protein-fimbriae interactions.
Structural and Evolutionary Inference from Molecular Variation in
Neisseria
Porins
Authors: Derrick Jeremy P. , Urwin Rachel , Suker Janet , Feavers Ian M. , Maiden Martin C. J.
Published: December 2019

The porin proteins of the pathogenic
Neisseria
species,
Neisseria gonorrhoeae
and
Neisseria meningitidis
, are important as serotyping antigens, putative vaccine components, and for their proposed role in the intracellular colonization of humans. A three-dimensional structural homology model for
Neisseria
porins was generated from
Escherichia coli
porin structures and
N. meningitidis
PorA and PorB sequences. The
Neisseria
sequences were readily assembled into the 16-strand β-barrel fold characteristic of porins, despite relatively low sequence identity with the
Escherichia
proteins. The model provided information on the spatial relationships of variable regions of peptide sequences in the PorA and PorB trimers and insights relevant to the use of these proteins in vaccines. The nucleotide sequences of the porin genes from a number of other
Neisseria
species were obtained by PCR direct sequencing and from GenBank. Alignment and analysis of all available
Neisseria
porin sequences by use of the structurally conserved regions derived from the PorA and PorB structural models resulted in the recovery of an improved phylogenetic signal. Phylogenetic analyses were consistent with an important role for horizontal genetic exchange in the emergence of different porin classes and confirmed the close evolutionary relationships of the porins from
N. meningitidis
,
N. gonorrhoeae
,
Neisseria lactamica
, and
Neisseria polysaccharea
. Only members of this group contained three conserved lysine residues which form a potential GTP binding site implicated in pathogenesis. The model placed these residues on the inside of the pore, in close proximity, consistent with their role in regulating pore function when inserted into host cells.
Identification of Virulence Genes of
Helicobacter pylori
by Random Insertion Mutagenesis
Authors: Bijlsma J. J. E. , Vandenbroucke-Grauls C. M. J. E. , Phadnis S. H. , Kusters J. G.
Published: December 2019

The complete genome of the gram-negative bacterial pathogen
Helicobacter pylori
, an important etiological agent of gastroduodenal disease in humans, has recently been published. This sequence revealed that the putative products of roughly one-third of the open reading frames (ORFs) have no significant homology to any known proteins. To be able to analyze the functions of all ORFs, we constructed an integration plasmid for
H. pylori
and used it to generate a random mutant library in this organism. This integration plasmid, designated pBCα3, integrated randomly into the chromosome of
H. pylori
. To test the capacity of this library to identify virulence genes, subsets of this library were screened for urease-negative mutants and for nonmotile mutants. Three urease-negative mutants in a subset of 1,251 mutants (0.25%) and 5 nonmotile mutants in a subset of 180 mutants (2.7%) were identified. Analysis of the disrupted ORFs in the urease-negative mutants revealed that two had disruptions of genes of the urease locus,
ureB
and
ureI
, and the third had a disruption of a unrelated gene; a homologue of
deaD
, which encodes an RNA helicase. Analysis of the disrupted ORFs in the nonmotile mutants revealed one ORF encoding a homologue of the paralyzed flagellar protein, previously shown to be involved in motility in
Campylobacter jejuni
. The other four ORFs have not been implicated in motility before. Based on these data, we concluded that we have generated a random insertion library in
H. pylori
that allows for the functional identification of genes in
H. pylori
.
Humoral Immune Responses toNeisseria meningitidisin Children
Authors: Pollard Andrew J. , Galassini Rachel , Rouppe van der Voort Eileene M. , Booy Robert , Langford Paul , Nadel Simon , Ison Catherine , Kroll J. Simon , Poolman Jan , Levin Michael
Published: December 2019

An understanding of the nature of immunity to serogroup B meningococci in childhood is necessary in order to establish the reasons for poor responses to candidate vaccines in infancy. We sought to examine the nature of humoral immune responses following infection in relation to age. Serum bactericidal activity was poor in children under 12 months of age despite recent infection withNeisseria meningitidis. The highest levels of bactericidal activity were seen in children over 10 years of age. However, infants produced levels of total immunoglobulin G (IgG) and IgG subclass antibodies similar to those in older children in a meningococcal enzyme-linked immunosorbent assay. Most antibody was of the IgG1 and IgG3 subclasses. This striking age dependency of bactericidal antibody response following infection is not apparently due to failure of class switching in infants but might be due to qualitative differences in antibody specificity or affinity.
In Vivo Analysis of Secreted Aspartyl Proteinase Expression in Human Oral Candidiasis
Authors: Naglik Julian R. , Newport George , White Theodore C. , Fernandes-Naglik Lynette L. , Greenspan John S. , Greenspan Deborah , Sweet Simon P. , Challacombe Stephen J. , Agabian Nina
Published: December 2019

Secreted aspartyl proteinases are putative virulence factors inCandidainfections.Candida albicanspossesses at least nine members of aSAPgene family, all of which have been sequenced. Although the expression of theSAPgenes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detectC. albicansmRNA from whole saliva of patients with oralC. albicansinfection and those with asymptomaticCandidacarriage. The reverse transcription-PCR protocol was used to determine which of theSAP1toSAP7genes are expressed byC. albicansduring colonization and infection of the oral cavity.SAP2and theSAP4toSAP6subfamily were the predominant proteinase genes expressed in the oral cavities of bothCandidacarriers and patients with oral candidiasis;SAP4,SAP5, orSAP6mRNA was detected in all subjects.SAP1andSAP3transcripts were observed only in patients with oral candidiasis.SAP7mRNA expression, which has never been demonstrated under laboratory conditions, was detected in several of the patient samples. All sevenSAPgenes were simultaneously expressed in some patients with oral candidiasis. This is the first detailed study showing that theSAPgene family is expressed byC. albicansduring colonization and infection in humans and thatC. albicansinfection is associated with the differential expression of individualSAPgenes which may be involved in the pathogenesis of oral candidiasis.
Structural Requirements of the Major Protective Antibody toHaemophilus influenzaeType b
Authors: Hougs Lotte , Juul Lars , Svejgaard Arne , Barington Torben
Published: December 2019

Protective antibodies to the important childhood pathogenHaemophilus influenzaetype b (Hib) are directed against the capsular polysaccharide (HibCP). Most of the antibody is encoded by a well-defined set of (“canonical”) immunoglobulin genes, including the VκA2 gene, and expresses an idiotypic marker (HibId-1). In comparison to noncanonical antibodies, the canonical antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats. Using site-directed mutagenesis, we here characterize canonical HibCP antibodies expressed as antigen-binding fragments (Fabs) inEscherichia coli, define amino acids involved in antigen binding and idiotype expression, and propose a three-dimensional structure for the variable domains. We found that canonical Fabs, unlike a noncanonical Fab, bound effectively to HibCP in the absence of somatic mutations. Nevertheless, pronounced mutation-based affinity maturation was demonstrated in vivo. An almost perfect correlation was found between unmutated gene segments that mediated binding in vitro and those encoding canonical HibCP antibodies in vivo. Thus, the VκA2a gene could be replaced by the A2c gene but not by the highly homologous sister gene, A18b, corresponding to the demonstrated usage of A2c but not of A18b in vivo. Similarly, only Jκ1 and Jκ3, which predominate in the response in vivo, were able to facilitate binding in vitro. These findings suggest that the restricted immunoglobulin gene usage in HibCP antibodies reflects strict structural demands ensuring relatively high affinity prior to somatic mutations—requirements met by only a limited spectrum of immunoglobulin gene combinations.
Towards aTaenia soliumCysticercosis Vaccine: an Epitope Shared byTaenia crassicepsandTaenia soliumProtects Mice against Experimental Cysticercosis
Authors: Toledo Andrea , Larralde Carlos , Fragoso Gladis , Gevorkian Goar , Manoutcharian Karen , Hernández Marisela , Acero Gonzalo , Rosas Gabriela , López-Casillas Fernando , Garfias Carlos Kubli , Vázquez Ricardo , Terrazas Ignacio , Sciutto Edda
Published: December 2019

TheTaenia crassicepsrecombinant antigen KETc7 has been shown to be effective as a vaccine against experimental murine cysticercosis, a laboratory model used to test potentially promising molecules against porcineTaenia soliumcysticercosis. Based on the deduced amino acid sequence of this proline-rich polypeptide, three fragments, GK-1, GK-2, and GK-3, were chemically synthesized in linear form. Of the three peptides, only GK-1 induced sterile protection againstT. crassicepscysticercosis in 40 to 70% of BALB/cAnN male mice. GK-1 is an 18-amino-acid peptide which contains at least one B-cell epitope, as demonstrated by its ability to induce an antibody response to the peptide andT. crassicepsantigen without need of a carrier protein. Immunofluorescence studies revealed that anti-GK1 antibodies strongly react with the native protein in the tegument ofT. crassicepsand also with anatomical structures ofT. soliumeggs, oncospheres, cysticercus, and tapeworm. GK-1 also contains at least one T-cell epitope, capable of stimulating the proliferation of CD8+and to a lower extent CD4+T cells primed either with the free peptide orT. crassicepstotal antigen. The supernatant of the stimulated cells contained high levels of gamma interferon and low levels of interleukin-4. Similar results were obtained with T cells tested for intracellular cytokine production, an indication of the peptide’s capacity to induce an inflammatory response. The remarkable protection induced by GK-1 immunization, its physicochemical properties, and its presence in all developmental stages ofT. soliumpoint to this synthetic peptide as a strong candidate in the construction of a synthetic vaccine againstT. soliumpig cysticercosis.
Comparative Physical and Genetic Maps of the Virulence Plasmids of
Salmonella enterica
Serovars Typhimurium, Enteritidis, Choleraesuis, and Dublin
Authors: Chu Chishih , Hong Seau-Feng , Tsai Chingju , Lin Wen-Shiun , Liu Tsui-Ping , Ou Jonathan T.
Published: December 2019

Using fragment profiling, PCR, and Southern hybridization, we found that
Salmonella enterica
serovar Choleraesuis harbored virulence plasmids of various sizes, whereas serovars Typhimurium, Enteritidis, and Dublin carried a plasmid of a unique size. Also, the virulence plasmid of Typhimurium contained genes in the same order detected in the other three plasmids, all of which contained deletions.
Localization of
HArep
-Containing Genes on the Chromosome of
Porphyromonas gingivalis
W83
Authors: Lewis Janina P. , Macrina Francis L.
Published: December 2019

We have mapped a group of virulence genes of
Porphyromonas gingivalis
to a single large fragment of the genome. These genes (
rgpA
,
kgp
, and
hagA
) all contain a consensus repeat sequence (
HArep
).
rgpA
and
kgp
encode cysteine proteases with Arg-X and Lys-X specificity, respectively, and
hagA
encodes a hemagglutinin. Genomic DNA fragments separated by pulse-field gel electrophoresis were blotted and probed in order to localize the genes to a 0.25-Mb
Nhe
I fragment of the
P. gingivalis
W83 genome. Further hybridization analyses with single- and double-restriction digestion allowed us to generate a physical map of the fragment and determine the precise locations of the protease and hemagglutinin genes. In addition, we found an insertion-like sequence, IS
195
, near the ends of the 0.25-Mb
Nhe
I fragment. A similarly sized fragment carrying
HArep
sequences was also demonstrated in the
P. gingivalis
W12 and W50 genomes.
Characterization of the Importance of Polysaccharide Intercellular Adhesin/Hemagglutinin ofStaphylococcus epidermidisin the Pathogenesis of Biomaterial-Based Infection in a Mouse Foreign Body Infection Model
Authors: Rupp Mark E. , Ulphani Joseph S. , Fey Paul D. , Bartscht Katrin , Mack Dietrich
Published: December 2019

The production of biofilm is thought to be crucial in the pathogenesis of prosthetic-device infections caused byStaphylococcus epidermidis. An experimental animal model was used to assess the importance of biofilm production, which is mediated by polysaccharide intercellular adhesin/hemagglutinin (PIA/HA), in the pathogenesis of a biomaterial-based infection. Mice were inoculated along the length of a subcutaneously implanted intravenous catheter with either wild-typeS. epidermidis1457 or its isogenic PIA/HA-negative mutant. The wild-type strain was significantly more likely to cause a subcutaneous abscess than the mutant strain (P< 0.01) and was significantly less likely to be eradicated from the inoculation site by host defense (P< 0.05). In addition, the wild-type strain was found to adhere to the implanted catheters more abundantly than the PIA/HA-negative mutant (P< 0.05). The reliability of the adherence assay was assessed by scanning electron microscopy. To exclude contamination or spontaneous infection, bacterial strains recovered from the experimental animals were compared to inoculation strains by analysis of restriction fragment length polymorphism patterns by pulsed-field gel electrophoresis. In vitro binding of the wild-type strain and its isogenic mutant to a fibronectin-coated surface was similar. These results confirm the importance of biofilm production, mediated by PIA/HA, in the pathogenesis ofS. epidermidisexperimental foreign body infection.
The Cellular Form of Human Fibronectin as an Adhesion Target for the S Fimbriae of Meningitis-Associated
Escherichia coli
Authors: Sarén Anne , Virkola Ritva , Hacker Jörg , Korhonen Timo K.
Published: December 2019

The adhesion of the S fimbriae of meningitis-associated
Escherichia coli
O18ac:K1:H7 to the cellular and the plasma forms of human fibronectin was studied.
E. coli
HB101(pAZZ50) expressing the complete S-fimbria II gene cluster of
E. coli
O18 adhered to cellular fibronectin (cFn) on glass but not to plasma fibronectin (pFn). Adhesion to cFn was specifically inhibited by neuraminidase treatment of cFn as well as by incubation of the bacteria with sialyl-α2-3-lactose, a receptor analog of the S fimbriae. No significant adhesion to cFn or pFn was detected with
E. coli
HB101(pAZZ50-67) expressing S fimbriae lacking the SfaS lectin subunit. Strain HB101(pAZZ50) also adhered to a human fibroblast cell culture known to be rich in cFn, and the adhesion was specifically inhibited in the presence of polyclonal antibodies to cFn. The results show that the SfaS lectin of the S fimbriae mediates the adherence of meningitis-associated
E. coli
to sialyl oligosaccharide chains of cFn.
Gamma Interferon and Interleukin-10 Gene Expression in Synovial Tissues from Patients with Early Stages ofChlamydia-Associated Arthritis and Undifferentiated Oligoarthritis and from Healthy Volunteers
Authors: Kotake Shigeru , Schumacher H. Ralph , Arayssi Thurayya K. , Gérard Herve C. , Branigan Patrick J. , Hudson Alan P. , Yarboro Cheryl H. , Klippel John H. , Wilder Ronald L.
Published: December 2019

Genetically determined differences in interleukin-10 (IL-10) and gamma interferon (IFN-γ) responses in mice correlate with clearance ofChlamydiapneumonitis infection. We measured the synovial expression of IL-10 and IFN-γ and additional cytokine genes in patients who had recent-onsetChlamydia-associated arthritis (Chl-AA). IL-10 and IFN-γ mRNA were relatively abundant in recent-onset Chl-AA.
Molecular Characterization of Equine Isolates of
Streptococcus pneumoniae
: Natural Disruption of Genes Encoding the Virulence Factors Pneumolysin and Autolysin
Authors: Whatmore Adrian M. , King Samantha J. , Doherty Neil C. , Sturgeon Daniel , Chanter Neil , Dowson Christopher G.
Published: December 2019

Although often considered a strict human pathogen,
Streptococcus pneumoniae
has been reported to infect and cause pneumonia in horses, although the pathology appears restricted compared to that of human infections. Here we report on the molecular characterization of a group of
S. pneumoniae
isolates obtained from horses in England and Ireland. Despite being obtained from geographically distinct locations, the isolates were found to represent a tight clonal group, virtually identical to each other but genetically distinguishable from more than 120 divergent isolates of human
S. pneumoniae
. A comprehensive analysis of known pneumococcal virulence determinants was undertaken in an attempt to understand the pathogenicity of equine pneumococci. Surprisingly, equine isolates appear to lack activities associated with both the hemolytic cytotoxin pneumolysin, often considered a major virulence factor of pneumococci, and the major autolysin gene
lytA
, also considered an important virulence factor. In support of phenotypic data, molecular studies demonstrated a deletion of parts of the coding sequences of both
lytA
and
ply
genes in equine pneumococci. The implications of these findings for the evolution and pathogenicity of equine
S. pneumoniae
are discussed.
Environmental Modulation of Oral Treponeme Virulence in a Murine Model
Authors: Kesavalu Lakshmyya , Holt Stanley C. , Ebersole Jeffrey L.
Published: December 2019

This investigation examined the effects of environmental alteration on the virulence of the oral treponemesTreponema denticolaandTreponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth ofT. denticolaandT. pectinovorumas a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth ofT. denticolaorT. pectinovorumunder iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions.T. denticolainduced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast,T. pectinovorumvirulence was minimally affected by this in vivo treatment to increase iron availability.T. denticolavirulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with bothT. denticolaandT. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity ofT. denticolaandT. pectinovorumby environmental conditions which can be evaluated by using in vivo murine models.
Inhibition of Osteoblastic Cell Differentiation by Lipopolysaccharide Extract fromPorphyromonas gingivalis
Authors: Kadono Hiroyuki , Kido Jun-Ichi , Kataoka Masatoshi , Yamauchi Noriyuki , Nagata Toshihiko
Published: December 2019

Lipopolysaccharide fromPorphyromonas gingivalis(P-LPS), an important pathogenic bacterium, is closely associated with inflammatory destruction of periodontal tissues. P-LPS induces the release of cytokines and local factors from inflammatory cells, stimulates osteoclastic-cell differentiation, and causes alveolar bone resorption. However, the effect of P-LPS on osteoblastic-cell differentiation remains unclear. In this study, we investigated the effect of P-LPS extract prepared by the hot-phenol–water method, on the differentiation of primary fetal rat calvaria (RC) cells, which contain a subpopulation of osteoprogenitor cells, into osteoblastic cells. P-LPS extract significantly inhibited bone nodule (BN) formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker, in a dose-dependent manner (0 to 100 ng of P-LPS extract per ml). P-LPS extract (100 ng/ml) significantly decreased BN formation to 27% of the control value and inhibited ALPase activity to approximately 60% of the control level on days 10 to 21 but did not affect RC cell proliferation and viability. P-LPS extract time-dependently suppressed the expression of ALPase mRNA, with an inhibitory pattern similar to that of enzyme activity. The expression of mRNAs for osteocalcin and osteopontin, matrix proteins related to bone metabolism, was markedly suppressed by P-LPS extract. Furthermore, P-LPS extract increased the expression of mRNAs for CD14, LPS receptor, and interleukin-1β in RC cells. These results indicate that P-LPS inhibits osteoblastic-cell differentiation and suggest that LPS-induced bone resorption in periodontal disease may be mediated by effects on osteoblastic as well as osteoclastic cells.