bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examination of Early Interactions between
Haemophilus ducreyi
and Host Cells by Using Cocultured HaCaT Keratinocytes and Foreskin Fibroblasts
Authors: Zaretzky Franca R. , Kawula Thomas H. 
Published: December 2019

ABSTRACT

Haemophilus ducreyi
is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Keratinocytes are likely the first cell type encountered by
H. ducreyi
upon infection of human skin; thus, the interaction between
H. ducreyi
and keratinocytes is probably important for the ability of
H. ducreyi
to establish infection. We have used the HaCaT keratinocyte cell line grown in monolayers and in cocultures with HS27 fibroblasts to investigate
H. ducreyi
interactions with keratinocytes and the host-cell response to
H. ducreyi
infection. Using quantitative adherence and gentamicin protection assays, we determined that approximately 13% of
H. ducreyi
adhered to HaCaT cell monolayers, while only a small proportion (0.0052%) was intracellular. By transmission electron microscopy, we observed numerous
H. ducreyi
organisms adherent to but rarely within HaCaT cells cocultured with fibroblasts. Both live
H. ducreyi
and purified
H. ducreyi
lipooligosaccharide (LOS) induced significant interleukin 8 (IL-8) expression from HaCaT cell-HS27 cell cocultures. However, the level of IL-8 expression in response to LOS alone was not as pronounced.
H. ducreyi
LOS was a more potent inducer of IL-8 from cocultures than
Escherichia coli
lipopolysaccharide (LPS) at the same concentration, suggesting a unique effect of
H. ducreyi
LOS on cocultures. Neither live
H. ducreyi
nor purified
H. ducreyi
LOS or
E. coli
LPS induced tumor necrosis factor alpha expression from cocultures.
H. ducreyi
induced drastically different cytokine profiles from cocultures than from HS27 or HaCaT cells cultured separately. IL-8 expression by skin cells in response to
H. ducreyi
infection in vivo may be responsible for the massive influx of polymorphonuclear leukocytes and other inflammatory cells to the site of infection. This influx of inflammatory cells may be partly responsible for the tissue destruction characteristic of chancroid.
Functional Characterization of a Serine/Threonine Protein Kinase of
Pseudomonas aeruginosa
Authors: Motley S. Timothy , Lory Stephen 
Published: December 2019

ABSTRACT

Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in
Pseudomonas aeruginosa
which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The
ppkA
gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in
P. aeruginosa
, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.
Conservation and Accessibility of an Inner Core Lipopolysaccharide Epitope ofNeisseria meningitidis
Authors: Plested Joyce S. , Makepeace Katherine , Jennings Michael P. , Gidney Margaret Anne J. , Lacelle Suzanne , Brisson J.-R. , Cox Andrew D. , Martin Adele , Bird A. Graham , Tang Christoph M. , Mackinnon Fiona M. , Richards James C. , Moxon E. Richard 
Published: December 2019

ABSTRACTWe investigated the conservation and antibody accessibility of inner core epitopes ofNeisseria meningitidislipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with agalEmutant ofN. meningitidisH44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains.N. meningitidisstrains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the β-chain heptose (HepII) of the inner core. In contrast,N. meningitidisstrains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms ofN. meningitidisLPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused byN. meningitidis.
Complete DNA Sequence and Structural Analysis of the Enteropathogenic
Escherichia coli
Adherence Factor Plasmid
Authors: Tobe Toru , Hayashi Tetsuya , Han Chang-Gyun , Schoolnik Gary K. , Ohtsubo Eiichi , Sasakawa Chihiro 
Published: December 2019

ABSTRACT

The complete nucleotide sequence and organization of the enteropathogenic
Escherichia coli
(EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the
bfp
operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the
bfpTVW
(
perABC
) operon, composed of regulatory genes required for
bfp
transcription and also for transcriptional activation of the
eae
gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the
bfp
and
bfpTVW
(
perABC
) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic
E. coli
. Another ORF, located between the
bfp
and
bfpTVW
operons, showed high similarity with
trcA
, a
bfpT
-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.
Divergent Roles of Tumor Necrosis Factor and Platelet-Activating Factor in Endotoxin-Induced Release of Monocyte Chemoattractant Protein 1 and Macrophage Inflammatory Protein 1β in Chimpanzees
Authors: Dekkers Pascale E. P. , Levi Marcel , van Deventer Sander J. H. , van der Poll Tom 
Published: December 2019

ABSTRACTA platelet-activating factor receptor antagonist reduced the release of macrophage inflammatory protein 1β (MIP-1β) during endotoxemia in chimpanzees but did not influence the secretion of monocyte chemoattractant protein 1 (MCP-1). Anti-tumor necrosis factor alpha monoclonal antibody completely prevented MCP-1 release and simultaneously enhanced the secretion of MIP-1β. Levels of MIP-1β and MCP-1 release were differentially regulated during endotoxemia.
Treatment with Succinic Anhydride Improves the Immunogenicity ofShigella flexneriType 2a O-Specific Polysaccharide–Protein Conjugates in Mice
Authors: Pavliakova Danka , Chu Chiayung , Bystricky Slavomir , Tolson Nathaniel W. , Shiloach Joseph , Kaufman Jeanne B. , Bryla Dolores A. , Robbins John B. , Schneerson Rachel 
Published: December 2019

ABSTRACTSeroepidemiological data and a clinical trial with aShigella sonneiO-specific polysaccharide (O-SP)–Pseudomonas aeruginosarecombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9orrEPA, bound to the O-SP ofShigella flexneritype 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9served as a more immunogenic carrier thanrEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.
Pseudomonas aeruginosaInduces Type-III-Secretion-Mediated Apoptosis of Macrophages and Epithelial Cells
Authors: Hauser Alan R. , Engel Joanne N. 
Published: December 2019

ABSTRACTPseudomonas aeruginosais a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with theP. aeruginosastrain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates ofP. aeruginosainduced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which theP. aeruginosatype III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.
Dichotomy of Cytokine Profiles in Patients and High-Risk Healthy Subjects Exposed to Tuberculosis
Authors: Bhattacharyya S. , Singla R. , Dey A. B. , Prasad H. K. 
Published: December 2019

ABSTRACTTo better understand the role of cytokines in susceptible and resistant subjects exposed toMycobacterium tuberculosisinfection, intracellular gamma interferon (IFN-γ) and interleukin-4 (IL-4) in ex vivo peripheral blood-derived CD4+T cells were examined by flow cytometry. Of the 37 individuals examined, 20 had clinical evidence of pulmonary tuberculosis and showed acid-fast bacilli in the sputum. Other individuals in close contact with these patients showed no evidence of disease. Patients had a higher number of CD4+T cells expressing IFN-γ and IL-4 in unstimulated cultures compared to healthy subjects. Despite this, the ratio of IFN-γ+to IL-4+CD4+T cells was similar in both groups. The Th1 response seen in CD4+T cells in patients was also observed in the overall pattern of IFN-γ and IL-4 detected in control culture supernatants by enzyme-linked immunosorbent assay (ELISA). However, after in vitro stimulation of PBMC with heat-killedM. tuberculosisthere was a significant reduction in the percentage of IFN-γ+CD4+T cells (P< 0.001) in patients. This trend was reflected in the IFN-γ ELISA assay with supernatants derived from stimulated cultures. However, the accumulated levels of IFN-γ were higher than those for IL-4. The reduction of IFN-γ+CD4+T cells resulted in the dominance of IL-4+CD4+T cells in 13 patients (P< 0.05). The elevated levels of IL-4+CD4+T cells seen in patients may contribute to the downregulation of IFN-γ expression and the crucial effector function of CD4 T cells, leading to the persistence of disease and the immunopathology characteristically seen in patients. Preliminary data on the indicators of apoptosis in antigen-stimulated cultures in PBMC derived from patients are presented. Of the 17 high-risk healthy individuals examined, 11 differed in that, after mycobacterial-antigen stimulation, there was an enhancement in IFN-γ+CD4+T cells.
Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+T Cells of theMycobacterium lepraeMemory Repertoire
Authors: Mustafa Abu S. , Lundin Knut E. A. , Meloen Robert H. , Shinnick Thomas M. , Oftung Fredrik 
Published: December 2019

ABSTRACTBy using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to theMycobacterium lepraememory repertoire. A panel of HSP65 reactive CD4+T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killedM. lepraeand then tested for proliferative reactivity against overlapping peptides comprising both theM. lepraeandMycobacterium tuberculosisHSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between theM. tuberculosisandM. lepraeHSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) representedM. lepraeHSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from selfHLA-DRB1genes, whereas 3 peptides with sequences completely identical between theM. lepraeandM. tuberculosisHSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4+T cells which belong to the human memory T-cell repertoire againstM. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.
Salmonella typhimurium
Virulence Genes Are Induced upon Bacterial Invasion into Phagocytic and Nonphagocytic Cells
Authors: Pfeifer Cheryl G. , Marcus Sandra L. , Steele-Mortimer Olivia , Knodler Leigh A. , Finlay B. Brett 
Published: December 2019

ABSTRACT

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify
Salmonella typhimurium
genes that are induced inside
Salmonella
-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the
Salmonella
chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in
S. typhimurium
that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the
ssaR
gene, within
Salmonella
pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the
sopB/sigD
gene and the other in a downstream gene,
pipB
. The insertions within SPI-5 resulted in the attenuation of
S. typhimurium
in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the
S. typhimurium
chromosome,
iicA
(induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in
Salmonella
pathogenesis.
Construction and Characterization of
Moraxella catarrhalis
Mutants Defective in Expression of Transferrin Receptors
Authors: Luke Nicole R. , Campagnari Anthony A. 
Published: December 2019

ABSTRACT

We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, in
Moraxella catarrhalis
7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the
tbpA
mutant and the
tbpAB
mutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, the
ompB1
(
tbpB
) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the
tbpA
mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, the
ompB1
(
tbpB
) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the
M. catarrhalis
TbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by
M. catarrhalis
.
Enhanced Macrophage Resistance toPseudomonasExotoxin A Is Correlated with Decreased Expression of the Low-Density Lipoprotein Receptor-Related Protein
Authors: Laithwaite James E. , Benn Sally J. , Yamate Jyoji , FitzGerald David J. , LaMarre Jonathan 
Published: December 2019

ABSTRACTCellular intoxification by exotoxin A ofPseudomonas aeruginosa(PEA) begins when PEA binds to its cellular receptor, the low-density lipoprotein receptor-related protein (LRP). This receptor is particularly abundant on macrophages. We hypothesize here that inducible changes in cellular expression levels of the LRP represent an important mechanism by which macrophage susceptibility to PEA is regulated by the host. We have examined the effect of lipopolysaccharide (LPS) on LRP expression and PEA sensitivity in the macrophage-like cell line HS-P. Using a [3H]leucine incorporation assay to measure inhibition of protein synthesis, we have demonstrated that HS-P macrophages are highly sensitive to PEA and that PEA toxicity is decreased by the LRP antagonist receptor-associated protein. LPS pretreatment decreases HS-P PEA sensitivity in a time- and dose-dependent manner. The dose of toxin required to inhibit protein synthesis by 50% increased from 11.3 ± 1.2 ng/ml in untreated cells to 25.7 ± 2.0 ng/ml in cells treated with LPS. In pulse experiments, involving brief exposure to saturating concentrations of PEA, [3H]leucine incorporation was more than threefold higher in cells pretreated with LPS than in untreated macrophages. These changes in HS-P PEA sensitivity following LPS treatment were consistently associated with a fivefold decrease in HS-P LRP mRNA expression as measured by Northern blot analysis and a three-and-a-half-fold decrease in HS-P LRP-specific ligand internalization as determined by activated α2-macroglobulin internalization studies. These data demonstrate for the first time that modulation of LRP levels by extracellular signaling molecules can alter cellular PEA sensitivity.
Reactive Nitrogen and Oxygen Species Ameliorate Experimental Cryptosporidiosis in the Neonatal BALB/c Mouse Model
Authors: Leitch Gordon J. , He Qing 
Published: December 2019

ABSTRACTFour-day-old BALB/c mice were infected by the oral administration of 50,000Cryptosporidium parvumoocysts, and the resulting infection was scored histologically and by counting colonic oocysts. Infection occurred in the ileum and proximal colon (but not duodenum and jejunum), peaked on days 14 to 18, and was cleared between days 24 and 30. Nitric oxide (NO) appeared to play a protective role in this model as evidenced by the facts that plasma nitrite and nitrate levels increased during the period of peak parasitosis; immunohistochemically detected inducible nitric oxide synthase (iNOS) was increased in the ileum and colon enterocytes of infected animals; the NOS inhibitorl-N-iminoethyl lysine orN-nitro-l-arginine methyl ester (L-NAME) decreased the elevated plasma nitrite and nitrate levels while exacerbating the infection and increasing oocyst shedding; administration of a NO donor,S-nitroso-N-penicillamine, reduced oocyst and infection scores; and neonatal iNOS knockout mice exhibited a slightly longer infection than control animals. The oral administration of oocysts to L-NAME-treated BALB/c mice, but not control animals, between 24 and 40 days old resulted in the fecal excretion of oocysts 1 week later. Administration of the antioxidant ascorbic acid also exacerbated theC. parvuminfection, suggesting a protective role for reactive nitrogen and/or reactive oxygen compounds, while administration of the superoxide scavenger superoxide dismutase exacerbated the infection. Taken together these data suggest that both reactive nitrogen and reactive oxygen species play protective roles in experimental cryptosporidiosis.