bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proinflammatory and Proapoptotic Activities Associated withBordetella pertussisFilamentous Hemagglutinin
Authors: Abramson Tzvia , Kedem Hassya , Relman David A. 
Published: July 2002

ABSTRACTFilamentous hemagglutinin (FHA) is a dominant cell surface-associatedBordetella pertussisadhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other actvities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-typeB. pertussisresulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-α), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-α monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-α receptor 1 inhibited FHA-associated apoptosis by 47.7% (P= 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in theB. pertussis-host interaction.
Vacuolating Cytotoxin of
Helicobacter pylori
Induces Apoptosis in the Human Gastric Epithelial Cell Line AGS
Authors: Kuck Dirk , Kolmerer Bernhard , Iking-Konert Christof , Krammer Peter H. , Stremmel Wolfgang , Rudi Jochen 
Published: July 2002

ABSTRACT

Helicobacter pylori
induces cell death by apoptosis. However, the apoptosis-inducing factor is still unknown. The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS. The supernatant from the
vacA
wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not. That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA. Furthermore, a recombinant VacA produced in
Escherichia coli
was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation. These findings demonstrate that the vacuolating cytototoxin of
H. pylori
is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.
Deficient Humoral Responses Underlie Susceptibility toToxoplasma gondiiin CD4-Deficient Mice
Authors: Johnson Lawrence L. , Sayles Peter C. 
Published: July 2002

ABSTRACTResistance to infection withToxoplasma gondiiwas studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain ofT. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroralT. gondiiinfection with cysts and impairs their ability to be successfully vaccinated. CD8+T cells from blood or spleens ofToxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-γ in vitro was moderately depressed, and levels ofToxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration ofToxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8+T cells and IFN-γ, which are known to be critical for resistance, CD4+cells also contribute significantly to protection against chronicT. gondiiinfections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.
Cytotoxic Activities of
Leptospira interrogans
Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells
Authors: Lee Seoung Hoon , Kim Sangduk , Park Seung Chul , Kim Min Ja 
Published: July 2002

ABSTRACT

Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans.
Leptospira
sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of
Leptospira interrogans
serovar lai, a homologue of
L. borgpetersenii
sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19–28, 2000).
Escherichia coli
lysate harboring the
sphH
showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the
Leptospira
hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of
Leptospira
SphH was due to the pore formation on mammalian cell membranes.
Increased Activation and Oligoclonality of Peripheral CD8+T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis
Authors: Manfras Burkhard J. , Reuter Stefan , Wendland Thomas , Kern Peter 
Published: July 2002

ABSTRACTAlveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+and CD8+T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens andEchinococcus multilocularisvesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+but not CD4+T cells, suggesting a persistent antigenic drive for CD8+T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+T cells in AE.
Bacteriophage φYeO3-12, Specific for
Yersinia enterocolitica
Serotype O:3, Is Related to Coliphages T3 and T7
Authors: Pajunen Maria , Kiljunen Saija , Skurnik Mikael 
Published: July 2002

ABSTRACT

Bacteriophage φYeO3-12 is a lytic phage of
Yersinia enterocolitica
serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-
l
-altropyranose. A one-step growth curve of φYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy φYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole φYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of φYeO3-12. φYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that φYeO3-12 is the first close relative of phage T3 to be described.
Anaerobic Toluene Catabolism of
Thauera aromatica
: the
bbs
Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate
Authors: Leuthner Birgitta , Heider Johann 
Published: July 2002

ABSTRACT

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent β-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in β oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of
bbs
(beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the β-oxidation pathway of benzylsuccinate. Two of the genes,
bbsE
and
bbsF
, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown
Thauera aromatica
. The
bbsG
gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in
Escherichia coli
and detection of enzyme activity. The further enzymes of the pathway are probably encoded by
bbsH
(enoyl-CoA hydratase),
bbsCD
(3-hydroxyacyl-CoA dehydrogenase), and
bbsB
(3-oxoacyl-CoA thiolase). The operon contains two additional genes,
bbsA
and
bbsI
, for which no obvious function could be derived. The
bbs
operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the
bbsA
gene. This represents the first known promoter site in
Thauera
spp.
Production of an Endo-β-N-Acetylglucosaminidase Activity Mediates Growth ofEnterococcus faecalison a High-Mannose-Type Glycoprotein
Authors: Roberts Gretta , Tarelli Edward , Homer Karen A. , Philpott-Howard John , Beighton David 
Published: July 2002

ABSTRACTEnterococcus faecalisis associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man5- to Man9-GlcNAc2), to support growth ofE. faecaliswas investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linkedN-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action ofStreptomyces plicatusendo-β-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only oneN-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity inE. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans byE. faecalismay play a role in the survival and persistence of this nosocomial pathogen in vivo.
The
gaf
Fimbrial Gene Cluster of
Escherichia coli
Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Authors: Tanskanen Jarna , Saarela Sirkku , Tankka Sanna , Kalkkinen Nisse , Rhen Mikael , Korhonen Timo K. , Westerlund-Wikström Benita 
Published: July 2002

ABSTRACT

The GafD lectin of the G (F17) fimbriae of diarrhea-associated
Escherichia coli
was overexpressed and purified from the periplasm of
E. coli
by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named ΔGafD. Expression of
gafD
from the cloned
gaf
gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of ΔGafD. The peptide was also detected in the periplasm of the wild-type
E. coli
strain from which the
gaf
gene cluster originally was cloned. We expressed
gafD
fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of ΔGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using
gafD
indicated that ΔGafD was processed from GafD and is not a primary translation product. The ΔGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant
E. coli
strain and in the binding of [
14
C]ΔGafD to GlcNAc-agarose. ΔGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when
gafD
is expressed alone or from the
gaf
gene cluster.
Analyses of
mrp
Genes during
Myxococcus xanthus
Development
Authors: Sun Hong , Shi Wenyuan 
Published: July 2002

ABSTRACT

Myxococcus xanthus
is a gram-negative soil bacterium that undergoes development under starvation conditions. Our previous study identified a new genetic locus,
mrp
, which is required for both fruiting body formation and sporulation. The locus encodes two transcripts:
mrpAB
, which consists of a histidine kinase and an NtrC-like response regulator, and
mrpC
, a cyclic AMP receptor protein family transcription activator. In this study, we used genetic and biochemical analyses to investigate the possible interactions between the
mrp
genes and other known developmental genes and events. These studies show that the
mrp
genes possibly function after A-signaling and (p)ppGpp but before C-signaling and that they regulate various early and late developmental genes and events.
Postsegregational Killing Mediated by the P1 Phage “Addiction Module”
phd-doc
Requires the
Escherichia coli
Programmed Cell Death System
mazEF
Authors: Hazan Ronen , Sat Boaz , Reches Myriam , Engelberg-Kulka Hanna 
Published: July 2002

ABSTRACT

“Addiction modules” consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module
phd-doc
, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The
Escherichia coli
chromosomal addiction module analogue,
mazEF
, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1
phd-doc
module depends on the presence of the
E. coli mazEF
system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by
phd-doc
, protein synthesis of
E. coli
is inhibited. Based on our findings, we suggest the existence of a coupling between the
phd-doc
and
mazEF
systems.
The DevT Protein Stimulates Synthesis of FruA, a Signal Transduction Protein Required for Fruiting Body Morphogenesis in
Myxococcus xanthus
Authors: Boysen Anders , Ellehauge Eva , Julien Bryan , Søgaard-Andersen Lotte 
Published: July 2002

ABSTRACT

Fruiting body formation in
Myxococcus xanthus
involves three morphologic stages—rippling, aggregation, and sporulation—all of which are induced by the cell surface-associated C-signal. We analyzed the function of the DevT protein, a novel component in the C-signal response pathway. A mutant carrying an in-frame deletion in the
devT
gene displays delayed aggregation and a cell autonomous sporulation defect, whereas it remains rippling proficient. To further define the function of DevT, the methylation pattern of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein homologue, was examined in the Δ
devT
mutant, and we found that DevT is required for methylation of FrzCD during development. Specifically, DevT was found to be required for the C-signal-dependent methylation of FrzCD. The Δ
devT
mutant produced wild-type levels of C-signal. However, accumulation of the FruA response regulator protein, which is essential for the execution of the C-signal-dependent responses, was reduced in the Δ
devT
mutant. The DevT protein was found to stimulate the developmentally activated transcription of the
fruA
gene. Epistasis analyses indicate that DevT acts independently of the A- and E-signals to stimulate
fruA
transcription. These findings suggest that the developmental defects of the Δ
devT
mutant are associated with a lack of FruA to ensure a proper response to the C-signal during the aggregation and sporulation stages of development.
Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Ebola and Marburg Viruses Using Recombinant Nucleoproteins
Authors: Saijo Masayuki , Niikura Masahiro , Morikawa Shigeru , Ksiazek Thomas G. , Meyer Richard F. , Peters Clarence J. , Kurane Ichiro 
Published: July 2002

ABSTRACT

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione
S
-transferase-tagged recombinant proteins in an
Escherichia coli
system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.
Identification of New Repetitive Element in
Leptospira interrogans
Serovar Copenhageni and Its Application to PCR-Based Differentiation of
Leptospira
Serogroups
Authors: Barocchi Michele A. , Ko Albert I. , Ferrer Suzana Ramos , Faria Marcos Tucunduva , Reis Mitermayer Galvão , Riley Lee W. 
Published: July 2002

ABSTRACT

A new repetitive DNA element was identified in an isolate of
Leptospira interrogans
serovar copenhageni from a patient in Salvador, Brazil. A
Sau
3A genomic library from this strain was constructed and screened for repetitive DNA elements. An insert of 438 bp (Rep1) from one library clone hybridized to multiple chromosomal DNA fragments resolved electrophoretically after digestion with
Bam
HI,
Hin
dIII, and
Mfe
I. A single oligonucleotide primer, designated iRepl, was designed to generate multiple PCR amplicons of various electrophoretic mobilities in a PCR typing method. The method distinguished strains belonging to the eight pathogenic and three saprophytic species of the genus
Leptospira
. Clinical isolates obtained during urban epidemics between 1996 and 1998 in Salvador, Brazil, were analyzed by this PCR method. Although the iRep1 primer was unable to discriminate strains among
L. interrogans
serovar copenhageni isolates, it was able to differentiate strains belonging to different species and serogroups of
Leptospira
identified in Salvador. This PCR-based method may provide a faster and less expensive alternative to serologic tests used in reference laboratories.