bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Levels of Soluble CD30 in Plasma of Patients withPlasmodium falciparumMalaria
Authors: Kemp Kåre , Kurtzhals Jørgen A. L. , Akanmori Bartholomew D. , Adabayeri Victoria , Goka Bamenla Q. , Behr Charlotte , Hviid Lars
Published: July 2002

Levels of soluble CD30 (sCD30) in serum were elevated in patients withPlasmodium falciparummalaria but showed decline following treatment. The levels of sCD30 in serum were correlated significantly with the expression of gamma interferon by peripheral T cells. These data suggest that CD30+cells are upregulated during a malaria attack and that they may play a regulating role at the site of inflammation.
Identification of Attenuated
Yersinia pseudotuberculosis
Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis
Authors: Mecsas Joan , Bilis Inna , Falkow Stanley
Published: July 2002

Yersinia pseudotuberculosis
localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13
Y. pseudotuberculosis
mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type
Y. pseudotuberculosis
. Several other mutations were in genes that had not previously been associated with growth in a host, including
cls, ksgA
, and
sufl
. In addition, using Y.
pseudotuberculosis
strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.
Correlation of Susceptibility of Immature Mice to Fungal Infection (Blastomycosis) and Effector Cell Function
Authors: Ganer Ari , Brummer Elmer , Stevens David A.
Published: July 2002

Immature mice are highly susceptible to blastomycosis, which is similar to other mycoses and has parallels in humans. The murine susceptibility is noteworthy in that it persists beyond the development of resistance to other, nonfungal pathogens and the maturation of most immune functions. As the susceptibility to blastomycosis appeared to be related to an early event after infection, primary effector cell function was studied. We found that peritoneal inflammatory cells, enriched for neutrophils, from immature (3-week-old) mice killed nonphagocytizableBlastomyces dermatitidiscells less (25%) than did cells from mature (8-week) mice (70%) (P< 0.01), a defect intrinsic to the neutrophils. This correlated with an impaired immature cell oxidative burst. Killing of phagocytizableCandida albicanswas not significantly different, 73 versus 87%. Thioglycolate-elicited cells were more impaired; killing ofB. dermatitidiswas insignificant, and killing ofC. albicanswas more impaired in immature (16% killing) than in mature (45%) cells (P< 0.02). Peripheral blood neutrophils from mature animals killedB. dermatitidis(41%) more than did those from immature animals (10%) (P< 0.02);C. albicanswas killed efficiently by both. Resting or activated peritoneal macrophages from both types of animals showed no differences inB. dermatitidiskilling. These results suggest that the susceptibility of immature mice is related at least in part to the depressed capacity of their neutrophils to killB. dermatitidis.
Development of an In Vivo Model for Study of Intestinal Invasion bySalmonella entericain Chickens
Authors: Aabo S. , Christensen J. P. , Chadfield M. S. , Carstensen B. , Jensen T. K. , Bisgaard M. , Olsen J. E.
Published: July 2002

An in vivo loop test model for the investigation of the invasiveness ofSalmonella entericain chickens was developed. Ten jejunal loops were made in 10- to 12-week-old Lohman Brown chickens under isofluoran anaesthesia.Salmonellaat 5.0 × 107CFU was inoculated into each loop and left for 2 h, followed by a 1-h incubation with gentamicin in order to kill noninvading bacteria. After euthanasia,Salmonellainvasiveness was measured as tissue-associated counts relative to a reference strain. The ability ofSalmonellainvasion was 1 log10CFU higher per 42-mm2mucosal tissue in the anterior than in the posterior part of jejunum. A statistically significant (P< 0.001) sixfold difference in invasiveness was observed between a wild-typeS. entericaserotype Typhimurium strain and the correspondinginvHmutant. The model was shown to be able to show small differences in invasive capability and allows for comparison of strains tested in different animals, provided that the same reference strain is present in all animals.
Reduced Th1, but Not Th2, Cytokine Production by Lymphocytes after In Vivo Exposure of Healthy Subjects to Endotoxin
Authors: Lauw Fanny N. , ten Hove Tessa , Dekkers Pascale E. P. , de Jonge Evert , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced capacity of monocytes to produce proinflammatory cytokines upon restimulation in vitro. To determine whether LPS exposure induces a change in lymphocyte cytokine production and whether this results in a shift in the T-helper 1 (Th1)/Th2 balance, whole blood obtained from seven healthy subjects before and after an intravenous injection of LPS (4 ng/kg) was stimulated in vitro with the T-cell stimulus anti-CD3/CD28 or staphylococcal enterotoxin B. Whole-blood production of the Th1 cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) was markedly reduced at 3 and 6 h, while the production of the Th2 cytokines IL-4 and IL-5 was not influenced or was slightly increased. The IFN-γ/IL-4 ratio was strongly decreased at 6 h. Serum obtained after LPS exposure could slightly inhibit the release of IFN-γ but increased IL-4 production during stimulation of blood drawn from subjects not previously exposed to LPS. Normal serum also inhibited IFN-γ production, albeit to a lesser extent. LPS exposure influences lymphocyte cytokine production, resulting in a shift toward a Th2 cytokine response, an effect that may be mediated in part by soluble factors present in serum after LPS administration in vivo.
Synergistic Epithelial Responses to Endotoxin and a Naturally Occurring Muramyl Peptide
Authors: Flak Tod A. , Heiss Linda N. , Engle Jacquelyn T. , Goldman William E.
Published: July 2002

We have investigated the synergistic interactions of a naturally occurring peptidoglycan fragment (muramyl peptide) and bacterial endotoxin in the induction of inflammatory processes within respiratory epithelial cells, at the levels of both signal transduction events and ultimate cellular metabolic effects. The source of the muramyl peptide isBordetella pertussis, the causative agent of the respiratory disease pertussis. During log-phase growth,B. pertussisreleases the muramyl peptide tracheal cytotoxin (TCT), which has the structureN- acetylglucosaminyl - 1,6 - anhydro -N- acetylmuramyl - (l) - alanyl - γ - (d) - glutamyl -meso- diaminopimelyl - (d) - alanine, equivalent to a monomeric subunit of gram-negative bacterial peptidoglycan. When applied to hamster trachea epithelial (HTE) cells, TCT and endotoxin were found to be highly synergistic in the induction of interleukin-1α (IL-1α), type II (inducible) nitric oxide synthase (iNOS), nitric oxide production, and inhibition of DNA synthesis. Neither molecule alone significantly triggered these responses. The serine/threonine protein kinase inhibitor H7 blocked induction of both IL-1α and iNOS. More selective inhibitors of protein kinase C, cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase were not capable of blocking the effects of TCT and endotoxin, suggesting that the H7-inhibited component in this pathway is not among the commonly described kinase targets of H7. Treatment of HTE cells with exogenous IL-1 reproduced the induction of iNOS and DNA synthesis inhibition caused by TCT and endotoxin. H7 was not capable of interfering with effects caused by exogenous IL-1, implying that the H7-sensitive step in the pathway is upstream of IL-1 protein production. Similar assays with the phorbol ester phorbol myristate acetate indicate that it could effectively synergize with endotoxin but not with TCT, suggesting that TCT and endotoxin induce different signal transduction events that combine synergistically. The synergy observed with TCT and endotoxin in epithelial cells is significantly different from their interaction with other cell types, revealing a unique inflammatory response by epithelial cells to these natural bacterial products.
Heterologous Expression ofTrypanosoma cruzi trans-Sialidase inLeishmania majorEnhances Virulence
Authors: Belen Carrillo M. , Gao Wenda , Herrera Macario , Alroy Joseph , Moore Jeffrey B. , Beverley Stephen M. , Pereira Miercio A.
Published: July 2002

Earlier studies showed that mice primed for a few hours with thetrans-sialidase (TS) ofTrypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection. These studies suggest that TS affects parasite virulence independent of antigenic stimulation. Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite. We tested this hypothesis by expressing heterologous TS inLeishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product α2-3-linked sialic acid.Leishmaniacells transfected with aT. cruziTS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu. TS expression did not affectL. majorbinding to and entry into cultured macrophages or its tropism for macrophage infection in vivo. However, TS-expressingL. majorexhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro- and microscopic examination of cutaneous lesions. Several genetic tests proved that the enhanced virulence was directly attributable to TS expression. The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage onT. cruziand transgenicL. major. These data suggest that heterologous expression ofT. cruzivirulence factors inLeishmaniamay provide a new approach for dissecting their function in vivo.
Detection of 23 Immunogenic Pneumococcal Proteins Using Convalescent-Phase Serum
Authors: Zysk Gregor , Bongaerts Roger J. M. , ten Thoren Elke , Bethe Gesina , Hakenbeck Regine , Heinz Hans-Peter
Published: July 2002

A genomic expression library of
Streptococcus pneumoniae
was screened with a convalescent-phase serum for immunoreactive proteins. Six known and 17 unknown pneumococcal proteins were detected. Five of the known proteins were surface-located virulence factors, and eight of the unknown proteins were putative membrane proteins.
Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 byChlamydia pneumoniaein Human Endothelial Cells
Authors: Molestina Robert E. , Miller Richard D. , Lentsch Alex B. , Ramirez Julio A. , Summersgill James T.
Published: July 2002

Infection withChlamydia pneumoniae, a causative agent of acute and chronic respiratory diseases, has recently been implicated as a potential risk factor in atherosclerosis. Atherosclerotic lesions are characterized by monocyte infiltration, which may be regulated by the chemokine monocyte chemotactic protein 1 (MCP-1). We have previously shown thatC. pneumoniaeinfection stimulates MCP-1 production in human endothelial cells, an event which may be specific to this species ofChlamydia, sinceChlamydia trachomatisinfection fails to induce this response. To examine the underlying mechanisms by whichC. pneumoniaeinfection induces MCP-1 production in endothelial cells, the present study investigated the role of transcription factor NF-κB in MCP-1 mRNA expression. Human umbilical vein endothelial cells (HUVEC) were infected with the coronary isolateC. pneumoniaeA-03 or withC. trachomatisL2, and MCP-1 mRNA expression was assessed after different periods of infection by reverse transcription-PCR. Expression of MCP-1 mRNA inC. pneumoniae-infected HUVEC was significantly elevated as early as 1 h postinfection and increased dramatically by 12 and 24 h compared to baseline controls. Nuclear translocation of NF-κB occurred by 30 min of infection, as determined by electrophoretic mobility shift assays and immunofluorescence staining. Treatment ofC. pneumoniae-infected HUVEC with parthenolide, a specific inhibitor of NF-κB activation, suppressed MCP-1 mRNA expression. In contrast, infection withC. trachomatisL2 did not induce MCP-1 mRNA in infected HUVEC and failed to activate NF-κB. Results from this study demonstrate a requirement for NF-κB activation in stimulation of MCP-1 gene expression byC. pneumoniaein human endothelial cells. Furthermore, the data suggest that, within the genusChlamydia, functionally distinct signaling pathways leading to NF-κB activation are utilized byC. pneumoniaein endothelial cells during infection.
Adaptive Immunity againstListeria monocytogenesin the Absence of Type I Tumor Necrosis Factor Receptor p55
Authors: White Douglas W. , Badovinac Vladimir P. , Fan Xin , Harty John T.
Published: July 2002

Tumor necrosis factor (TNF) and the type I TNF receptor (TNFRI), p55, are critical for resistance against primary infections with the intracellular bacterial pathogenListeria monocytogenes. Importantly, however, susceptibility to primary listeriosis in cytokine-deficient mice does not preclude the development or expression of effective adaptive immunity against virulentL. monocytogenes. We used TNFRI−/−mice to study adaptive antilisterial immunity in the absence of interactions between TNF and TNFRI. Our experiments indicate that TNFRI−/−mice survive and clear high-dose challenges with an attenuated strain ofL. monocytogenesthat is incapable of cell-to-cell spread. Furthermore, TNFRI−/−mice immunized with attenuatedL. monocytogenesgo on to develop potent adaptive immunity to subsequent high-dose challenges with virulentL. monocytogenes. Interestingly, CD8+T-cell depletion in vivo inhibits immunity toL. monocytogenesin the spleen but not in the liver of TNFRI−/−mice. The adaptive immune response in these animals is characterized by activation of listeriolysin O-specific CD8+T cells, which are capable of transferring antilisterial immunity to naive wild-type C57BL/6 host mice. These experiments demonstrate the development and expression of potent CD8+T-cell-mediated antilisterial immunity in the absence of TNFRI.
Role of Monocytes in Experimental
Staphylococcus aureus
Endocarditis
Authors: Veltrop Marcel H. A. M. , Bancsi Maurice J. L. M. F. , Bertina Rogier M. , Thompson Jan
Published: July 2002

In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein.
Staphylococcus aureus
is a frequently isolated bacterium from patients with BE. We therefore investigated whether
S. aureus
can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of
S. aureus
infection on vegetational TFA. In vitro experiments showed that adherent
S. aureus
induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo
S. aureus
led to very high bacterial numbers in the vegetations and a significant increase of their weight. However, TFA of infected vegetations was the same as of sterile ones. This may be due to the high bacteria to monocyte ratio as well as bacterium-induced monocyte damage. Teicoplanin treatment of infected rabbits reduced bacterial numbers in the blood and in the vegetations. Two-day treatment resulted in an increase of vegetational TFA, but after four-day treatment vegetational TFA dropped, most probably due to a suboptimal bacterium/monocyte ratio.
S. aureus
endocarditis in etoposide (Vepesid)-treated rabbits, leading to a selective monocytopenia, caused a rapid death of the animals. In these rabbits no vegetations were found at all. We conclude that, like
Streptococcus sanguis
and
Staphylococcus epidermidis
,
S. aureus
is able to induce TFA in fibrin-adherent blood monocytes. In addition, monocytes have a protective effect during the course of
S. aureus
endocarditis.
Surface-Expressed Mig Protein ProtectsStreptococcus dysgalactiaeagainst Phagocytosis by Bovine Neutrophils
Authors: Song Xin-Ming , Perez-Casal Jose , Bolton Alexandra , Potter Andrew A.
Published: July 2002

Themiggene ofStreptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, α2-macroglobulin (α2-M) and immunoglobulin G (IgG). In this study, themiggene from oneS. dysgalactiaeisolate was cloned and expressed inEscherichia coli. The IgG receptor region encoded bymigwas conserved in 16S. dysgalactiaestrains. An isogenicmigmutant was constructed by allele replacement mutagenesis of the wild-type gene inS. dysgalactiae. The IgG-binding activity was lost in themigmutant strain, whereas the α2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than themigmutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein ofS. dysgalactiaeplays a role in virulence of the bacteria by binding to the plasma protein α2-M or IgG and thus preventing phagocytosis by bovine PMNs.
Susceptibilities ofCryptococcus neoformansStrains to Platelet Binding In Vivo and to the Fungicidal Activity of Thrombin-Induced Platelet Microbicidal Proteins In Vitro
Authors: Nail S. , Robert R. , Dromer F. , Marot-Leblond A. , Senet J. M.
Published: July 2002

In this study we investigated the interactions between capsular and acapsular strains ofCryptococcus neoformansand blood platelets. In vivo microscopic observation of blood samples from mice inoculated withC. neoformansyeast cells demonstrated that encapsulated and nonencapsulated yeast cells disappeared quickly from the bloodstream and that platelets were attached solely to yeast cells of the nonencapsulated strains. In vitro we observed that only the acapsular strains were susceptible to the fungicidal activity of thrombin-induced platelet microbicidal proteins.
In Vitro Investigation of Host Resistance toToxoplasma gondiiInfection in Microglia of BALB/c and CBA/Ca Mice
Authors: Freund Yvonne R. , Zaveri Naunihal T. , Javitz Harold S.
Published: July 2002

Toxoplasmic encephalitis (TE) is a life-threatening disease of immunocompromised individuals and has increased in prevalence as a consequence of AIDS. TE has been modeled in inbred mice, with CBA/Ca mice being susceptible and BALB/c mice resistant to the development of TE. To better understand the innate mechanisms in the brain that play a role in resistance to TE, nitric oxide (NO)-dependent and NO-independent mechanisms were examined in microglia from BALB/c and CBA/Ca mice and correlated with the ability of these cells to inhibitToxoplasma gondiireplication. These parameters were measured 48 h after stimulation with lipopolysaccharide (LPS) gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or combinations of these inducers inT. gondii-infected microglia isolated from newborn mice. CBA/Ca microglia consistently produced less NO than did BALB/c microglia after stimulation with LPS or with IFN-γ plus TNF-α, and they inhibitedT. gondiireplication significantly less than did BALB/c microglia. Cells of both strains treated with IFN-γ alone significantly inhibited uracil incorporation byT. gondii, andNG-monomethyl-l-arginine (NMMA) treatment did not reverse this effect. In cells treated with IFN-γ in combination with other inducers, NMMA treatment resulted in only partial recovery ofT. gondiireplication. This IFN-γ-dependent inhibition of replication was not due to generation of reactive oxygen species or to increased tryptophan degradation. These data suggest that NO production and an IFN-γ-dependent mechanism contribute to the inhibition ofT. gondiireplication after in vitro stimulation with IFN-γ plus TNF-α or with LPS. Differences in NO production but not in IFN-γ-dependent inhibition ofT. gondiireplication were observed between CBA/Ca and BALB/c microglia.
Interference between Host Resistance toListeria monocytogenesInfection and Ovalbumin-Induced Allergic Responses in Mice
Authors: Mizuki Daisuke , Miura Tomisato , Sasaki Sanae , Mizuki Mayuko , Madarame Hiroo , Nakane Akio
Published: July 2002

Listeria monocytogenespromotes the induction of the T-helper 1 (Th1) cell response, while ovalbumin (OVA) induces a Th2 cell response and allergic reactions, such as airway hyperreactivity and immunoglobulin E (IgE) production. When mice were immunized with OVA on day 7 afterL. monocytogenesinfection, eosinophilia in bronchoalveolar lavage and the production of total IgE, OVA-specific IgE, interleukin-4 (IL-4), and IL-5 in the circulation were markedly suppressed. Cytokine responses, including IL-4, IL-5, IL-10, IL-13, and gamma interferon, to OVA were decreased in the spleen cell cultures obtained from OVA-immunized mice that had been infected withL. monocytogenes. Conversely, when OVA-immunized mice were infected withL. monocytogenes, conversion from the nonlethal infection to the lethal infection occurred. Host resistance toL. monocytogenesinfection in OVA-immunized mice was enhanced by the administration of anti–IL-10 monoclonal antibody. The present study indicates that striking interference is observed between Th1-inducingL. monocytogenesinfection and Th2-driven OVA-induced airway hyperreactivity.
Regulation of Matrix Metalloproteinase Expression in Endothelial Cells by Heat-Inactivated
Streptococcus pneumoniae
Authors: Michel Uwe , Zobotke Rita , Mäder Michael , Nau Roland
Published: July 2002

Matrix metalloproteinases (MMPs) may contribute to an impaired endothelial layer in several diseases. We examined the effect of heat-inactivated
Streptococcus pneumoniae
R6 on MMP-2 and MMP-9 release by cultured aortic and brain capillary endothelial cells. Treatment with heat-inactivated
S. pneumoniae
caused an increased release of MMP-2 by both cell types.
Transcriptional Regulation of Divergent Capsule Biosynthesis and Transport Operon Promoters in Serogroup B
Neisseria meningitidis
Authors: Tzeng Yih-Ling , Swartley John S. , Miller Yoon K. , Nisbet Rachel E. , Liu Li-Jun , Ahn Jay H. , Stephens David S.
Published: July 2002

The clinically important serogroups B, C, Y, and W-135 of
Neisseria meningitidis
produce sialic acid capsules that are critical in pathogenesis. In each of these serogroups, the capsule transport (
ctrABCD
) and capsule biosynthesis (
synABCD
) operons are divergently transcribed from putative promoters located in a 134-bp intergenic region (J. S. Swartley, J. H. Ahn, L. J. Liu, C. M. Kahler, and D. S. Stephens, J. Bacteriol. 178:4052–4059, 1996). In this study we further assessed the role of the intergenic sequence in the transcriptional regulation of the sialic acid capsules of
N. meningitidis
. Insertional mutagenesis or deletions of the 134-bp sequence in the serogroup B meningococcal strain NMB resulted in a marked reduction or elimination of
ctrABCD
and
synABCD
transcription, with a concomitant loss of encapsulation. Chromosomal transcriptional
lacZ-ermC
reporter fusions of
syn
and
ctr
promoters were constructed through allelic exchange. Using these constructs, both operons were found to be constitutively transcribed in meningococci, the biosynthesis operon about fourfold higher than the transport operon. Both promoters showed increased activity during stationary-phase growth. In addition to the promoters, a 70-bp 5′ untranslated region (UTR) upstream of
synA
was found to have a direct repeat and an inverted repeat that overlapped three putative integration host factor binding sites. Mutation of this 70-bp UTR and of the direct repeat upregulated both
syn
and
ctr
transcription. Regulation through the
synA
UTR was absent in a K1
Escherichia coli
strain that produces identical capsular polysaccharide, implicating species-specific regulation. Meningococcal sialic acid capsule expression is initiated by divergent promoters in a 134-bp intergenic region, is repressed at the transcriptional level by the 5′ UTR of
synA
, is increased during stationary-phase growth, and shows species-specific regulation. Transcriptional regulation is another important control point for sialic capsule expression in
N. meningitidis
.
By Releasing ADP,Acanthamoeba castellaniiCauses an Increase in the Cytosolic Free Calcium Concentration and Apoptosis in Wish Cells
Authors: Mattana A. , Tozzi M. G. , Costa M. , Delogu G. , Fiori P. L. , Cappuccinelli P.
Published: July 2002

The role played by soluble molecules that may participate in acanthamoebal cytopathogenicity has yet to be fully characterized. We demonstrate here thatAcanthamoeba castellaniitrophozoites constitutively release ADP in the medium. Cell-free supernatants prepared fromA. castellanii, by interaction with specific P2y2purinoceptors expressed on the Wish cell membrane, caused a biphasic rise in [Ca2+]i, extensive cell membrane blebbing, cytoskeletal disorganization, and the breakdown of nuclei. Cell damage induced by amoebic supernatants was blocked by the P2y2inhibitor Suramin. The same results were found in Wish cells exposed to purified ADP. These findings suggest that pathogenic free-livingA. castellaniimay have a cytopathic effect on human epithelial cells through ADP release, by a process that begins with a rise of cytosolic free-calcium concentration, and culminates in apoptosis.
Development of a Gene Inactivation System for
Bacteroides forsythus
: Construction and Characterization of a BspA Mutant
Authors: Honma Kiyonobu , Kuramitsu Howard K. , Genco Robert J. , Sharma Ashu
Published: July 2002

Bacteroides forsythus
is a gram-negative anaerobic bacterium associated with periodontitis. The
bspA
gene encoding a cell surface associated leucine-rich repeat protein (BspA) involved in adhesion to fibronectin and fibrinogen was recently cloned from this bacterium in our laboratory. We now describe the construction of a BspA-defective mutant of
B. forsythus
. This is the first report describing the generation of a specific gene knockout mutant of
B. forsythus
, and this procedure should be useful in establishing the identity of virulence-associated factors in these organisms.
Mycoplasma pneumoniae
P1 Type 1- and Type 2-Specific Sequences within the P1 Cytadhesin Gene of Individual Strains
Authors: Dorigo-Zetsma J. Wendelien , Wilbrink Berry , Dankert Jacob , Zaat Sebastian A. J.
Published: July 2002

Mycoplasma pneumoniae
strains traditionally are divided into two types, based on sequence variation in the P1 gene. Recently, however, we have identified 8 P1 subtypes by restriction fragment length polymorphism analysis. In the present study the P1 gene sequences of three P1 type 1 and two P1 type 2
M. pneumoniae
strains were analyzed. A new P1 gene sequence in a type 1 strain with partial similarity to a recently reported variable region in the P1 gene of an
M. pneumoniae
type 2 strain (T. Kenri, R. Taniguchi, Y. Sasaki, N. Okazaki, M. Narita, K. Izumikawa, M. Umetsu, and T.Sasaki, Infect. Immun. 67:4557–4562, 1999) was identified. In addition, the P1 gene of the type 1 strain contained another region with nucleotide polymorphisms identical to a stretch in the P1 gene of one of our type 2 strains. These findings indicate that recombination between sequences specific for P1 type 1 and type 2 had occurred and that P1 type 1 and type 2 hybrid sequences can be present within the P1 gene of an individual strain. Identical or nearly identical variable P1 gene sequences were present in several repetitive regions outside the P1 gene locus in the genome of
M. pneumoniae
strain M129, implying recombination as a mechanism for generation of the P1 gene variation. Additionally, in the P1 gene sequences of four of the five strains studied, single-nucleotide polymorphisms different from the previously reported P1 type 1 and 2 characteristic sequences were identified. The polymorphic sites are candidate targets for genotyping of
M. pneumoniae
by direct sequencing of amplicons from clinical specimens.
Distinct Proinflammatory Host Responses to
Neisseria gonorrhoeae
Infection in Immortalized Human Cervical and Vaginal Epithelial Cells
Authors: Fichorova Raina Nakova , Desai Pragnya Jasvantrai , Gibson Frank C. , Genco Caroline Attardo
Published: July 2002

In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated,
N. gonorrhoeae
strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by
N. gonorrhoeae
.
Essential Role of Interleukin-12 (IL-12) and IL-18 for Gamma Interferon Production Induced by Listeriolysin O in Mouse Spleen Cells
Authors: Nomura Takamasa , Kawamura Ikuo , Tsuchiya Kohsuke , Kohda Chikara , Baba Hisashi , Ito Yutaka , Kimoto Terumi , Watanabe Isao , Mitsuyama Masao
Published: July 2002

The mechanism of gamma interferon (IFN-γ) production induced by listeriolysin O (LLO), a cytolytic virulence factor ofListeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant ofL. monocytogeneswas capable of inducing a high level of IFN-γ when its cytolytic activity was blocked by cholesterol treatment. The IFN-γ-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b+cells resulted in a profound decrease in IFN-γ production in response to LLO stimulation. Negative selection also suggested the contribution of DX5+cells in IFN-γ production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b+fraction of spleen cells was unchanged. There was no change in the expression of the IFN-γ-inducing cytokine genes in the CD11b−fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-γ production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-γ after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor ofL. monocytogenes, is capable of inducing IFN-γ from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.
Mapping and Sequencing of the Canine NRAMP1 Gene and Identification of Mutations in Leishmaniasis-Susceptible Dogs
Authors: Altet Laura , Francino Olga , Solano-Gallego Laia , Renier Corinne , Sánchez Armand
Published: July 2002

The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, includingLeishmania. Our aims were to sequence and map the canineSlc11a1gene and to identify mutations that may be associated with resistance or susceptibility toLeishmaniainfection. The canineSlc11a1gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected withLeishmania infantumto search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility.
Helicobacter pyloriDoes Not Require Lewis X or Lewis Y Expression To Colonize C3H/HeJ mice
Authors: Takata Tohru , El-Omar Emad , Camorlinga Margarita , Thompson Stuart A. , Minohara Yutaka , Ernst Peter B. , Blaser Martin J.
Published: July 2002

Helicobacter pyloristrains frequently express Lewis X (Lex) and/or Leyon their cell surfaces as constituents of the O antigens of their lipopolysaccharide molecules. To assess the effect of Lexand Leyexpression on the ability ofH. pylorito colonize the mouse stomach and to adhere to epithelial cells, isogenic mutants were created in whichfucT1alone orfucT1andfucT2, which encode the fucosyl transferases necessary for Lexand Leyexpression, were deleted. C3H/HeJ mice were experimentally challenged with either wild-type 26695H. pylorior its isogenic mutants. All strains, whether passaged in the laboratory or recovered after mouse passage, colonized the mice well and without consistent differences. During colonization by the mutants, there was no reversion to wild type. Similarly, adherence to AGS and KatoIII cells was unaffected by the mutations. Together, these findings indicate that Le expression is not necessary for mouse gastric colonization or forH. pyloriadherence to epithelial cells.
Expression of the Neutral Protease Gene from a Thermophilic
Bacillus
sp. BT1 Strain in
Bacillus subtilis
and Its Natural Host: Identification of a Functional Promoter
Authors: Večerek Branislav , Venema Gerard
Published: July 2002

The expression of the neutral protease gene (
npr
) from the thermophilic
Bacillus
sp. BT1 strain was studied in its natural host and in mesophilic
Bacillus subtilis
. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5′ to the gene. In contrast, in heterologous
B. subtilis
this thermophilic
npr
promoter does not function, and expression of the
npr
gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5′ to ORF X that is required for efficient expression of the
npr
gene in
Bacillus subtilis
as verified by primer extension, reverse transcription-PCR, and 5′ rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic
Bacillus
sp. BT1 strain are different from those used in
B. subtilis
.
Self-Assembly of the
Agrobacterium tumefaciens
VirB11 Traffic ATPase
Authors: Rashkova Svetlana , Zhou Xue-Rong , Chen Jun , Christie Peter J.
Published: July 2002

The
Agrobacterium tumefaciens
VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of λ cI repressor protein formed dimers, as shown by immunity of
Escherichia coli
to λ superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type
A. tumefaciens
cells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized
A. tumefaciens
cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5′ and 3′ halves of
virB11
. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (ΔGKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.
Coactivation of the RpoS-Dependent
proP
P2 Promoter by Fis and Cyclic AMP Receptor Protein
Authors: McLeod Sarah M. , Xu Jimin , Johnson Reid C.
Published: July 2002

The
Escherichia coli proP
P2 promoter, which directs the expression of an integral membrane transporter of proline, glycine betaine, and other osmoprotecting compounds, is induced upon entry into stationary phase to protect cells from osmotic shock. Transcription from the P2 promoter is completely dependent on RpoS (ς
38
) and Fis. Fis activates transcription by binding to a site centered at −41, which overlaps the promoter, where it makes a specific contact with the C-terminal domain of the α subunit of RNA polymerase (α-CTD). We show here that Fis and cyclic AMP (cAMP) receptor protein (CRP)-cAMP collaborate to activate transcription synergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding to a site centered at −121.5, but CRP without Fis provides little activation. The contribution by CRP requires the correct helical phasing of the CRP site and a functional activation region 1 on CRP. We provide evidence that coactivation is achieved by Fis and CRP independently contacting each of the two α-CTDs. Efficient transcription in vitro requires that both activators must be preincubated with the DNA prior to addition of RNA polymerase.
oriT
-Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of the
Sinorhizobium meliloti
pExo Megaplasmid Replicator Region
Authors: Chain Patrick S. G. , Hernandez-Lucas Ismael , Golding Brian , Finan Turlough M.
Published: July 2002

We have developed a procedure to directly clone large fragments from the genome of the soil bacterium
Sinorhizobium meliloti
. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (
oriT
) together with a plasmid replication origin capable of replicating large clones in
Escherichia coli
but not in the target organism. Supplying transfer genes in trans specifically transfers the
oriT
-flanked region, and in this process, site-specific recombination at the
oriT
sites results in a plasmid carrying the flanked region of interest that can replicate in
E. coli
from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the
oriT
of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the
S. meliloti
pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium
Agrobacterium tumefaciens
. The nucleotide sequence of this fragment revealed a replicator region including homologs of the
repA
,
repB
, and
repC
genes from other
Rhizobiaceae
, which encode proteins involved in replication and segregation of plasmids in many organisms.
The Amino Terminus of
Salmonella enterica
Serovar Typhimurium ς
54
Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA
Authors: Kelly Mary T. , Hoover Timothy R.
Published: July 2002

Transcription initiation by the ς
54
-RNA polymerase holoenzyme requires an enhancer-binding protein that is thought to contact ς
54
to activate transcription. To identify potential enhancer-binding protein contact sites in ς
54
, we compared the abilities of wild-type and truncated forms of
Salmonella enterica
serovar Typhimurium ς
54
to interact with the enhancer-binding protein DctD in a chemical cross-linking assay. Removal of two regions in the amino-terminal portion of ς
54
, residues 57 to 105 and residues 144 to 179, prevented cross-linking, but removal of either region alone did not. In addition, deletion of 56 amino-terminal residues of ς
54
(region I) reduced the affinity of the protein for a fork junction DNA probe.
Functional Expression in
Escherichia coli
and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in
Helicobacter pylori
Authors: Bina Jim , Bains Manjeet , Hancock Robert E. W.
Published: July 2002

HopE is one of the smallest members of a family of 31 outer membrane proteins in
Helicobacter pylori
and has been shown to function as a porin. In this study it was cloned into
Escherichia coli
where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from
E. coli
reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from
H. pylori
. A model of the membrane topology of HopE was constructed and indicated that this protein formed a β-barrel with 16 transmembrane amphipathic β-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane β-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.
Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene,
rrlA
, of the Extreme Thermophile
Thermus thermophilus
IB-21
Authors: Gregory Steven T. , Cate Jamie H. D. , Dahlberg Albert E.
Published: July 2002

Spontaneous, erythromycin-resistant mutants of
Thermus thermophilus
IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of
Thermus
.
Disulfide Bond Formation in Secreton Component PulK Provides a Possible Explanation for the Role of DsbA in Pullulanase Secretion
Authors: Pugsley Anthony P. , Bayan Nicolas , Sauvonnet Nathalie
Published: July 2002

When expressed in
Escherichia coli
, the 15
Klebsiella oxytoca pul
genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these
pul
genes, efficient pullulanase secretion also requires the host
dsbA
gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a
dsbA
mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a
dsbA
mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.
Identification and Characterization of
gsp65
, an Organic Hydroperoxide Resistance (
ohr
) Gene Encoding a General Stress Protein in
Enterococcus faecalis
Authors: Rincé Alain , Giard Jean-Christophe , Pichereau Vianney , Flahaut Sigrid , Auffray Yanick
Published: July 2002

The
Enterococcus faecalis
general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the
gsp65
product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of
gsp65
gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and
tert
-butylhydroperoxide (tBOOH). A
gsp65
mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.
Gene Structures and Regulation of the Alkane Hydroxylase Complex in
Acinetobacter
sp. Strain M-1
Authors: Tani Akio , Ishige Takeru , Sakai Yasuyoshi , Kato Nobuo
Published: July 2002

In the long-chain
n
-alkane degrader
Acinetobacter
sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two
n
-alkane hydroxylase-encoding genes in response to the chain length of
n
-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.