bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endotoxin-Neutralizing Protein Protects against Endotoxin-Induced Endothelial Barrier Dysfunction
Authors: Bannerman Douglas D. , Fitzpatrick Michael J. , Anderson Dell Y. , Bhattacharjee Apurba K. , Novitsky Thomas J. , Hasday Jeffrey D. , Cross Alan S. , Goldblum Simeon E. 
Published: December 2019

ABSTRACTBacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived fromLimulusantilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (≥10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of ≥10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.
Human Mannose-Binding Protein Inhibits Infection of HeLa Cells byChlamydia trachomatis
Authors: Swanson Albertina F. , Ezekowitz R. Alan B. , Lee Amy , Kuo Cho-chou 
Published: December 2019

ABSTRACTThe role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection byChlamydia trachomatis(C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu),Chlamydia pneumoniae(AR-39), andChlamydia psittaci(6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L2was not affected by supplementation with complement or addition of an L2-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein ofC. trachomatisL2, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.
A Recombinant Live Attenuated Strain of
Vibrio cholerae
Induces Immunity against Tetanus Toxin and
Bordetella pertussis
Tracheal Colonization Factor
Authors: Chen Inês , Finn Theresa M. , Yanqing Liu , Guoming Qi , Rappuoli Rino , Pizza Mariagrazia 
Published: December 2019

ABSTRACT

An attenuated strain of
Vibrio cholerae
was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from
Bordetella pertussis
(Tcf). In vitro, high levels of protein were obtained when the
Escherichia coli nirB
promoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type
B. pertussis
. These data are in agreement with the putative role of Tcf in
Bordetella
tracheal colonization. In conclusion, we have demonstrated that
V. cholerae
may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.
Electrotransformation and Expression of Bacterial Genes Encoding Hygromycin Phosphotransferase and β-Galactosidase in the Pathogenic Fungus
Histoplasma capsulatum
Authors: Woods Jon P. , Heinecke Elizabeth L. , Goldman William E. 
Published: December 2019

ABSTRACT

We developed an efficient electrotransformation system for the pathogenic fungus
Histoplasma capsulatum
and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant
Escherichia coli
genes,
hph
and
lacZ
. Linearized DNA and plasmids containing
Histoplasma
telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable
URA5
marker (native
Histoplasma
gene or a heterologous
Podospora anserina
gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the
E. coli hph
gene flanked by
Aspergillus nidulans
promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in
H. capsulatum
, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing
Histoplasma URA5
with
E. coli lacZ
, resulting in expression of functional β-galactosidase in
H. capsulatum
. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of
H. capsulatum
, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.
Bordetella pertussisFilamentous Hemagglutinin Enhances the Immunogenicity of Liposome-Delivered Antigen Administered Intranasally
Authors: Poulain-Godefroy Odile , Mielcarek Nathalie , Ivanoff Nathalie , Remoué Franck , Schacht Anne-Marie , Phillips Nigel , Locht Camille , Capron André , Riveau Gilles 
Published: December 2019

ABSTRACTIn an attempt to increase the immunogenicity of mucosally delivered antigens, we incorporated theBordetella pertussisfilamentous hemagglutinin (FHA) adhesin into liposomes containing the glutathioneS-transferase ofSchistosoma mansoni(Sm28GST) as a model antigen. Outbred mice immunized twice intranasally with liposomes containing a constant suboptimal dose of Sm28GST and increasing doses of FHA produced anti-Sm28GST antibodies in a FHA dose-dependent manner. The addition of 3 μg of FHA to the liposomes induced more than 10-fold-higher anti-Sm28GST antibody titers, compared to those induced by liposomes without FHA. The presence of FHA did not alter the nature of the humoral immune response, and the sera contained anti-Sm28GST immunoglobulin G1 (IgG1), IgG2a, and IgG2b. However, anti-Sm28GST IgA was only detected when at least 3 μg of FHA was added to the preparation. These results show a promising potential for FHA to enhance the immunogenicity of mucosally administered antigens incorporated into liposomes.
Identification and Temperature Regulation of
Legionella pneumophila
Genes Involved in Type IV Pilus Biogenesis and Type II Protein Secretion
Authors: Liles Mark R. , Viswanathan V. K. , Cianciotto Nicholas P. 
Published: December 2019

ABSTRACT

Previously, we had isolated by transposon mutagenesis a
Legionella pneumophila
mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn
10
insertion, we found a locus that had a predicted protein product with strong similarity to PilB from
Pseudomonas aeruginosa
. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of
P. aeruginosa
PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when
L. pneumophila
was grown at 30°C as opposed to 37°C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the
pilB
locus was conserved within
L. pneumophila
serogroups and other
Legionella
species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.
Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells by
Rickettsia rickettsii
Authors: Sahni Sanjeev K. , Van Antwerp Daniel J. , Eremeeva Marina E. , Silverman David J. , Marder Victor J. , Sporn Lee Ann 
Published: December 2019

ABSTRACT

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB activation in cultured human umbilical vein endothelial cells consequent to infection with
Rickettsia rickettsii
, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786–2791, 1997). In the present study, we describe activation of NF-κB in a cell-free system, accomplished by addition of partially purified
R. rickettsii
to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of
R. rickettsii
organisms added. Antibody supershift assays using monospecific antisera against NF-κB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-κB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPγS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IκBα. This lack of IκBα involvement was supported by the finding that
R. rickettsii
can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB activation pathway wherein
R. rickettsii
may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
Evidence that Surface Proteins Sn14 and Sn16 ofSarcocystis neuronaMerozoites Are Involved in Infection and Immunity
Authors: Liang Fang Ting , Granstrom David E. , Zhao Xiao Min , Timoney John F. 
Published: December 2019

ABSTRACTSarcocystis neuronais the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity ofS. neuronaby an in vitro neutralization assay. A high correlation between immunoblot band pattern and neutralizing activity was detected. Two proteins, Sn14 and Sn16 (14 and 16 kDa, respectively), appeared to be important for in vitro infection. A combination of the results of surface protein labeling, immunoprecipitation, Western blotting, and trypsin digestion suggests that these molecules are surface proteins and may be useful components of a vaccine againstS. neuronainfection. AlthoughS. neuronais an obligate intracellular parasite, it is potentially a target for specific antibodies which may lyse merozoites via complement or inhibit their attachment and penetration to host cells.
Identification of the Aspartate-β-Semialdehyde Dehydrogenase Gene of
Legionella pneumophila
and Characterization of a Null Mutant
Authors: Harb Omar S. , Kwaik Yousef Abu 
Published: December 2019

ABSTRACT

The ability of
Legionella pneumophila
to cause Legionnaires’ disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where
L. pneumophila
survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of
L. pneumophila
to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of
L. pneumophila
by disruption of the aspartate-β-semialdehyde (
asd
) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the
asd
disruption since complementation of the mutant with the wild-type
asd
gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the
asd
gene may prove to be useful in the design of attenuated vaccines against Legionnaires’ disease.
Acquired Resistance ofEscherichia colito Complement Lysis by Binding of Glycophosphoinositol-Anchored Protectin (CD59)
Authors: Rautemaa Riina , Jarvis Gary A. , Marnila Pertti , Meri Seppo 
Published: December 2019

ABSTRACTProtectin (CD59) is a glycophosphoinsitol (GPI)-anchored defender of human cells against lysis by the membrane attack complex of complement. In this study, we examined whether protectin released from human cell membranes can incorporate into the surface of gram-negative bacteria. Analysis by using radiolabeled protectin, immunofluorescence, flow cytometry, and whole-cell enzyme-linked immunosorbent assay demonstrated that protectin bound to nonencapsulatedEscherichia coliEH237 (Re) and EH234 (Ra) in a calcium-dependent manner. The incorporation required the GPI-phospholipid moiety since no binding of a phospholipid-free soluble form of protectin was observed. Mg2+did not enhance the binding, and a polysialic acid capsule prevented it (strain IH3080 [O18:K1:H8]). Bound protectin inhibited the C5b-9 neoantigen expression on complement-treated bacteria. Protection against complement lysis was observed in both a colony counting assay and a bioluminescence assay, where viable EH234 bacteria expressing the luciferase gene emitted green light in the presence of the luciferine substrate. In general, two- to four-times-higher serum concentrations were needed to obtain 50% lysis of protectin-coated versus noncoated bacteria. The results indicate that protectin can incorporate in a functionally active form into the cell membranes of the two nonencapsulated deep roughE. colistrains studied.
Integrins αvβ3and α5β1Mediate Attachment of Lyme Disease Spirochetes to Human Cells
Authors: Coburn Jenifer , Magoun Loranne , Bodary Sarah C. , Leong John M. 
Published: December 2019

ABSTRACTBorrelia burgdorferi(sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin αIIbβ3was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferisensu stricto,B. garinii, andB. afzelii). Here we show thatB. burgdorferialso recognizes the widely expressed integrins αvβ3and α5β1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified αvβ3and α5β1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectiousB. burgdorferistrain N40 to purified αvβ3and α5β1was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to αvβ3was also shown by using a transfected cell line that expresses this receptor but not αIIbβ3. Attachment ofB. burgdorferiN40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α5β1and αvβ3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.
Cell-Contact-Stimulated Formation of Filamentous Appendages by
Salmonella typhimurium
Does Not Depend on the Type III Secretion System Encoded by
Salmonella
Pathogenicity Island 1
Authors: Reed Katharine A. , Clark M. Ann , Booth Trevor A. , Hueck Christoph J. , Miller Samuel I. , Hirst Barry H. , Jepson Mark A. 
Published: December 2019

ABSTRACT

The formation of filamentous appendages on
Salmonella typhimurium
has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717–724, 1994). We have examined the roles of cell contact and
Salmonella
pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of
S. typhimurium
strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type
S. typhimurium
bacteria adhering to both cultured epithelial cells and to murine Peyer’s patch follicle-associated epithelia. Wild-type
S. typhimurium
adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient
S. typhimurium
strains carrying mutations in components of SPI1 (
invA
,
invG
,
sspC
, and
prgH
) exhibited filamentous appendages similar to those on wild-type
S. typhimurium
when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an
S. typhimurium invG
mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on
S. typhimurium
strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.
Antibodies to RecombinantClostridium difficileToxins A and B Are an Effective Treatment and Prevent Relapse ofC. difficile-Associated Disease in a Hamster Model of Infection
Authors: Kink John A. , Williams Jim A. 
Published: December 2019

ABSTRACTClostridium difficilecauses antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treatC. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequentC. difficilereinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.