bluuebunny/crossref_metadata_2025_split huggingface dataset:
Translocated Intimin Receptors (Tir) of Shiga-Toxigenic
Escherichia coli
Isolates Belonging to Serogroups O26, O111, and O157 React with Sera from Patients with Hemolytic-Uremic Syndrome and Exhibit Marked Sequence Heterogeneity
Authors: Paton Adrienne W. , Manning Paul A. , Woodrow Matthew C. , Paton James C. 
Published: December 2019

ABSTRACT

The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic
Escherichia coli
(EPEC) and Shiga-toxigenic
E. coli
(STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and several other proteins necessary for generation of A/E lesions are encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). Reports of sequence heterogeneity and antigenic variation in the region of intimin believed to be responsible for receptor binding raise the possibility that the receptor itself is also heterogeneous. We have examined this by cloning and sequencing
tir
genes from three different STEC strains belonging to serogroups O26, O111, and O157. The deduced amino acid sequences for the Tir homologues from these strains varied markedly, exhibiting only 65.4, 80.2, and 56.7% identity, respectively, to that recently reported for EPEC Tir. STEC Tir is also highly immunogenic in humans. Western blots of
E. coli
DH5α expressing the various STEC
tir
genes cloned in pBluescript [but not
E. coli
DH5α(pBluescript)] reacted strongly with convalescent sera from patients with hemolytic-uremic syndrome (HUS) caused by known LEE-positive STEC. Moreover, no reaction was seen when the various clone lysates were probed with serum from a patient with HUS caused by a LEE-negative STEC or with serum from a healthy individual. Covariation of exposed epitopes on both intimin and Tir may be a means whereby STEC avoid host immune responses without compromising adhesin-receptor interaction.
Morphological Transition of
Paracoccidioides brasiliensis
Conidia to Yeast Cells: In Vivo Inhibition in Females
Authors: Aristizabal Beatriz H. , Clemons Karl V. , Stevens David A. , Restrepo Angela 
Published: December 2019

ABSTRACT

Clinical paracoccidioidomycosis is 13 times more common in men than in women. Estrogen inhibits the transition of mycelia or conidia (the saprophytic form of
Paracoccidoides brasiliensis
) to yeasts (the parasitic form) in vitro. Here, we show that, in male mice that were infected intranasally (mimicking natural infection) the transition of conidia in bronchoalveolar lavage fluids to intermediate forms and yeasts occurred over 24 to 96 h; CFU and yeasts (shown by histopathology) increased subsequently. In females, transition did not occur and infection cleared. These events in vivo are consistent with epidemiological and in vitro observations, suggesting that female hormones block transition and are responsible for resistance.
Genetic and Immunologic Analyses of PlpE, a Lipoprotein Important in Complement-Mediated Killing ofPasteurella haemolyticaSerotype 1
Authors: Pandher Karamjeet , Confer Anthony W. , Murphy George L. 
Published: December 2019

ABSTRACTPasteurella haemolyticaserotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies againstP. haemolyticaouter membrane proteins (OMPs) correlates statistically with resistance to experimentalP. haemolyticachallenge in cattle. Until now, specificP. haemolyticaOMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to anActinobacillus pleuropneumoniaelipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes ofP. haemolyticaexcept serotype 11. We found that intactP. haemolyticaand recombinantE. coliexpressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed inP. haemolyticaand assumes a similar surface-exposed conformation inE. coli. In complement-mediated killing assays, we observed a significant reduction in killing ofP. haemolyticawhen bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense againstP. haemolytica.
Sulfatide from the Pig Jejunum Brush Border Epithelial Cell Surface Is Involved in Binding of
Escherichia coli
Enterotoxin b
Authors: Rousset Élodie , Harel Josée , Dubreuil J. Daniel 
Published: December 2019

ABSTRACT

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of
Escherichia coli
heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3′-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO
4
)β1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277–288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.
Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection withListeria monocytogenesin Rats
Authors: Hirose Kenji , Suzuki Hirohiko , Nishimura Hitoshi , Mitani Akio , Washizu Junji , Matsuguchi Tetsuya , Yoshikai Yasunobu 
Published: December 2019

ABSTRACTExogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection withListeria monocytogenesin rats. In in vitro experiments, invasion ofL. monocytogenesinto IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation ofL. monocytogenesin vivo. The numbers of T-cell receptor (TCR) γδ+T cells, NKR.P1+cells, and CD3+CD8+αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection withL. monocytogenes.
Complete DNA Sequence and Detailed Analysis of the
Yersinia pestis
KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen
Authors: Lindler Luther E. , Plano Gregory V. , Burland Valerie , Mayhew George F. , Blattner Frederick R. 
Published: December 2019

ABSTRACT

Yersinia pestis
, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the
Y. pestis
-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of
Y. pestis
KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in
Y. pestis
. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the
parABS
bacteriophage P1 and P7 system.
Y. pestis
pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.
PseudomonasPyocyanin Increases Interleukin-8 Expression by Human Airway Epithelial Cells
Authors: Denning Gerene M. , Wollenweber Laura A. , Railsback Michelle A. , Cox Charles D. , Stoll Lynn L. , Britigan Bradley E. 
Published: December 2019

ABSTRACTPseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects ofP. aeruginosaare mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1α. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n -acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed inPseudomonas-associated lung disease.
Evaluation of Immunoglobulin A1 (IgA1) Protease and IgA1 Protease-Inhibitory Activity in Human Female Genital Infection withNeisseria gonorrhoeae
Authors: Hedges Spencer R. , Mayo Matthew S. , Kallman Lisa , Mestecky Jiri , Hook Edward W. , Russell Michael W. 
Published: December 2019

ABSTRACTImmunoglobulin A1 (IgA1) protease, an enzyme that selectively cleaves human IgA1, may be a virulence factor for pathogenic organisms such asNeisseria gonorrhoeae. Host protection from the effects of IgA1 protease includes antibody-mediated inhibition of IgA1 protease activity, and it is believed that the relative balance between IgA1 protease and inhibitory antibodies contributes to the pathogenesis of disease caused by IgA1 protease-producing organisms. We have examined the levels of these two opposing factors in genital tract secretions and sera from women with uncomplicated infection withN. gonorrhoeae. When IgA1 in cervical mucus was examined by Western blotting, no evidence of cleavage fragments characteristic of IgA1 protease activity was seen in gonococcus-infected or control patients. Cleavage fragments typical of IgA1 protease were detected, however, after the addition of exogenous IgA1 protease to cervical mucus. Degraded IgA1 was detected in some vaginal wash samples, but the fragment pattern was not typical of IgA1 protease activity. AllN. gonorrhoeaeisolates from the infected patients produced IgA1 protease in vitro. All but two serum samples and 16 of 65 cervical mucus samples displayed inhibitory activity against gonococcal IgA1 protease, but there was no significant difference in the level of inhibitory activity between gonococcus-infected and noninfected patients in either cervical mucus or serum. There was no difference in the levels of IgA1 protease-inhibitory activity in serum or cervical mucus collected from patients at recruitment and 2 weeks later. These results suggest that cleavage of IgA1 by gonococcal IgA1 protease within the lumen of the female lower genital tract is unlikely to be a significant factor in the pathogenesis of infections byN. gonorrhoeae.
Intranasal Immunization with Cytotoxic T-Lymphocyte Epitope Peptide and Mucosal Adjuvant Cholera Toxin: Selective Augmentation of Peptide-Presenting Dendritic Cells in Nasal Mucosa-Associated Lymphoid Tissue
Authors: Porgador Angel , Staats Herman F. , Itoh Yasushi , Kelsall Brian L. 
Published: December 2019

ABSTRACTWe previously reported that cholera toxin (CT) was required as a mucosal adjuvant for the induction of peptide-specific cytotoxic T lymphocytes (CTL) following intranasal immunization with CTL epitope peptides (A. Porgador et al., J. Immunol. 158:834–841, 1997). The present study was performed to identify the site and the antigen-presenting cell (APC) population responsible for the presentation of intranasally administered CTL epitope peptide immunogens and to determine whether CT directly affects antigen presentation by these APCs. For these experiments, C57BL/6 mice were intranasally immunized with the ovalbumin H-2Kb-restricted CTL epitope SIINFEKL with or without CT. Cells were then isolated from the cervical lymph nodes (CLN) and the nasal mucosa-associated lymphoid tissue (NALT) and tested for the ability to stimulate the B3Z T-cell hybridoma, which recognizes SIINFEKL in association with H-2Kb. Dendritic cell (DC)-enriched CLN cells from mice immunized with peptide and CT or peptide only could stimulate B3Z cells, while DC-depleted CLN cells from either group were unable to stimulate B3Z cells. NALT cells of mice immunized with peptide and CT, but not with peptide alone, were able to efficiently stimulate B3Z hybridomas. Depletion of N418-positive DC from these NALT cells resulted in significant reduction of B3Z activation. Our results indicate that DC are the APC responsible for the presentation of CTL epitope peptides following intranasal immunization and that CT augments the ability of dendritic cells in the NALT, but not in the draining CLN, to present CLT epitope peptides. This finding suggests that CT acts locally as a mucosal adjuvant and that NALT DC are the predominant APC involved with the induction of immunity after intranasal immunization with peptide immunogens and CT.
Identification of a 71-Kilodalton Surface-Associated Hsp70 Homologue in
Coxiella burnetii
Authors: Macellaro Anna , Tujulin Eva , Hjalmarsson Karin , Norlander Lena 
Published: December 2019

ABSTRACT

A
Coxiella burnetii
Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [
35
S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70,800. The −10 promoter sequence was shown to be identical with the consensus heat shock ς
32
promoter sequence. The base composition at the presumed −35 region revealed an
Eco
RI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in
Escherichia coli
as an intact protein. The
C. burnetii
71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g.,
Legionella pneumophila
and
Francisella tularensis
, as well as
E. coli
DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in
C. burnetii
cell fractions, using purified antibodies directed to the 71-kDa protein.
Selective Activation of
sar
Promoters with the Use of Green Fluorescent Protein Transcriptional Fusions as the Detection System in the Rabbit Endocarditis Model
Authors: Cheung A. L. , Nast Cynthia C. , Bayer A. S. 
Published: December 2019

ABSTRACT

The global regulatory locus
sar
is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individual
sar
promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless
gfp
UV
gene (a
gfp
derivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual
sar
promoters upstream of the
gfp
UV
reporter gene were then introduced into
Staphylococcus aureus
RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of the
gfp
UV
transcript driven by individual
sar
promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFP
UV
fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the
sar
P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of
sar
differed from that observed in vitro. Moreover, the individual
sar
promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the
sar
locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-
gfp
UV
constructs should be useful to characterize many aspects of
S. aureus
gene regulation in vivo.
Enteropathogenic
Escherichia coli
Virulence Genes Encoding Secreted Signalling Proteins Are Essential for Modulation of Caco-2 Cell Electrolyte Transport
Authors: Collington Georgina K. , Booth Ian W. , Donnenberg Michael S. , Kaper James B. , Knutton Stuart 
Published: December 2019

ABSTRACT

The pathophysiology of enteropathogenic
Escherichia coli
(EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current (
I
sc
) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this
I
sc
response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.
Invasion of Human Oral Epithelial Cells by
Prevotella intermedia
Authors: Dorn Brian R. , Leung K.-P. , Progulske-Fox Ann 
Published: December 2019

ABSTRACT

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of
Prevotella intermedia
, strain 17, was found to invade a human oral epithelial cell line (KB), whereas
P. intermedia
27, another clinical isolate, and
P. intermedia
25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of
P. intermedia
17. Antibodies raised against
P. intermedia
type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of
P. intermedia
can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.