bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of Chlorinated Benzenes and Toluenes by
Ralstonia
sp. Strain PS12
tecA
(Tetrachlorobenzene Dioxygenase) and
tecB
(Chlorobenzene Dihydrodiol Dehydrogenase) Gene Products
Authors: Pollmann Katrin , Beil Stefan , Pieper Dietmar H.
Published: July 2002

The
tecB
gene, located downstream of
tecA
and encoding tetrachlorobenzene dioxygenase, in
Ralstonia
sp. strain PS12 was cloned into
Escherichia coli
DH5α together with the
tecA
gene. The identity of the
tecB
gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.
Carbon Isotope Fractionation during Aerobic Biodegradation of Trichloroethene by
Burkholderia cepacia
G4: a Tool To Map Degradation Mechanisms
Authors: Barth Johannes A. C. , Slater Greg , Schüth Christoph , Bill Markus , Downey Angela , Larkin Mike , Kalin Robert M.
Published: July 2002

The strain
Burkholderia cepacia
G4 aerobically mineralized trichloroethene (TCE) to CO
2
over a time period of ∼20 h. Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD
540
s) in order to test whether isotope fractionation was consistent. The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD
540
s of 2.0, 1.1, and 0.6, respectively. ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 μmol h
−1
). While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille δ
13
C
VPDB
), they were 57.2, 39.6, and 17.0‰ between the initial and final TCE levels for the three experiments, in decreasing order of their OD
540
s. Despite these strong isotope shifts, we found a largely uniform isotope fractionation. The latter is expressed with a Rayleigh enrichment factor, ε, and was −18.2 when all experiments were grouped to a common point of 42.8% TCE remaining. Although, decreases of ε to −20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE. This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways.
Comparative and Prospective Study of Different Immune Parameters in Healthy Subjects at Risk for Tuberculosis and in Tuberculosis Patients
Authors: Portales-Pérez Diana P. , Baranda Lourdes , Layseca Esther , Fierro Nora Alma , de la Fuente Hortensia , Rosenstein Yvonne , González-Amaro Roberto
Published: July 2002

It has not been fully elucidated which of the components of the immune response againstMycobacterium tuberculosisis indicative of resistance or susceptibility. The aim of this study was to identify an immune parameter that could be indicative of either resistance or susceptibility toM. tuberculosisinfection. We prospectively studied (three determinations, at months 0, 8, and 12) 15 patients with chronic pulmonary tuberculosis and 42 healthy individuals with a recent and frequent contact with tuberculosis patients. Peripheral blood mononuclear cells were stimulated with a whole-protein extract or the 30-kDa antigen ofM. tuberculosisfor 6 days, and several immune parameters were determined. No consistent differences between tuberculosis patients and healthy controls were detected in most immune parameters studied, including the expression of different activation antigens, cytokine secretion, lymphocyte proliferation, and nitric oxide production. However, the synthesis of tumor necrosis factor alpha, the intracellular detection of gamma interferon, and the apoptosis of monocytes under certain culture conditions tended to show clear-cut differences in cells from patients and controls (P< 0.05 in all cases for most determinations). Nevertheless, when results were analyzed on an individual basis, it was evident that a significant degree of overlapping of values from patients and controls occurred for all parameters studied. We conclude that although the immune parameters tested do not allow the identification of individuals susceptible toM. tuberculosis, the specificity and sensitivity of some of them could be improved through future studies.
Early Nonspecific Immune Responses and Immunity to Blood-Stage NonlethalPlasmodium yoeliiMalaria
Authors: Choudhury Hasib R. , Sheikh Nadeem A. , Bancroft Gregory J. , Katz David R. , de Souza J. Brian
Published: July 2002

The early role of natural killer cells and gamma delta T cells in the development of protective immunity to the blood stage of nonlethalPlasmodium yoeliiinfection was studied. Splenic cytokine levels were measured 24 h after infection of natural killer cell-depleted immunodeficient and littermate mice or transiently T-cell-depleted normal mice. Splenic gamma interferon levels were significantly increased above background in immunodeficient and littermate mice 24 h after infection. Depletion of natural killer cells resulted in markedly depressed gamma interferon levels and poor control of parasitemia, particularly in severe combined immunodeficient mice. In the littermates, gamma interferon levels were partially reduced, but parasitemias were resolved normally. However, in athymic mice, natural killer cell depletion had no effect on gamma interferon production. Levels of tumor necrosis factor alpha were increased in all animals 24 h after infection, and responses were not affected by natural killer cell depletion. However, in T-cell-depleted animals, both gamma interferon and tumor necrosis factor alpha levels were decreased 24 h after infection, and depleted mice were unable to control their parasitemia. These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and gamma delta T cells contribute equally towards their production. The data also suggest that the subsequent resolution of infection requires early production of gamma interferon, which might act by switching on the appropriate T-helper-cell subsets and other essential parasitotoxic effector mechanisms.
Antigenic Variation ofAnaplasma marginaleby Expression of MSP2 Mosaics
Authors: Barbet Anthony F. , Lundgren Anna , Yi Jooyoung , Rurangirwa Fred R. , Palmer Guy H.
Published: July 2002

Anaplasma marginaleis a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. TheseEhrlichiaspecies have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome.A. marginaleand related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein ofA. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations ofA. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariablemsp2sequences to the promoter and polycistron.
Genetic and Functional Analysis of a PmrA-PmrB-Regulated Locus Necessary for Lipopolysaccharide Modification, Antimicrobial Peptide Resistance, and Oral Virulence of
Salmonella enterica
Serovar Typhimurium
Authors: Gunn John S. , Ryan Sara S. , Van Velkinburgh Jennifer C. , Ernst Robert K. , Miller Samuel I.
Published: July 2002

The two-component regulatory system PmrA-PmrB confers resistance of
Salmonella
spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), bactericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed
pmrE
and
pmrHFIJKLM
. Both
pmrE
and
pmrHFIJKLM
produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more positively charged lipopolysaccharide (LPS) and thus reduces cationic AP binding. Experiments were conducted to further analyze the regulation of the
pmrHFIJKLM
operon and the role of this operon and the surrounding genomic region in LPS modification and antimicrobial peptide resistance. The
pmrHFIJKLM
genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The
pmrHFIJKLM
promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated
pmrCAB
promoter. Construction of nonpolar mutations in the
pmrHFIJKLM
genes showed that all except
pmrM
were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (
pmrG
and
pmrD
) were not necessary for PM resistance, but
pmrD
was shown to be regulated by the PhoP-PhoQ regulatory system. BALB/c mice inoculated with
pmrA
and
pmrHFIJKLM
mutant strains demonstrated virulence attenuation when the strains were administered orally but not when they were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tissues.
Bacteroides fragilisNCTC9343 Produces at Least Three Distinct Capsular Polysaccharides: Cloning, Characterization, and Reassignment of Polysaccharide B and C Biosynthesis Loci
Authors: Coyne Michael J. , Kalka-Moll Wiltrud , Tzianabos Arthur O. , Kasper Dennis L. , Comstock Laurie E.
Published: July 2002

Bacteroides fragilisproduces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.
A Protective Glycosylphosphatidylinositol-Anchored Membrane Protein ofPlasmodium yoeliiTrophozoites and Merozoites Contains Two Epidermal Growth Factor-Like Domains
Authors: Burns James M. , Belk Carla C. , Dunn Patricia D.
Published: July 2002

Using sera from mice immunized and protected againstPlasmodium yoeliimalaria, we identified a novel blood-stage antigen gene,pypag-2. The 2.1-kbpypag-2cDNA contains a single open reading frame that encodes a 409-amino-acid protein with a predicted molecular mass of 46.8 kDa. Unlike many characterized plasmodial antigens, blocks of tandemly repeated amino acids are lacking in the pypAg-2 protein sequence. Recombinant pypAg-2, comprising the full-length protein minus the predicted N-terminal signal and C-terminal anchor sequences, was produced and used to raise a high-titer polyclonal rabbit antiserum. This antiserum was used to identify and characterize the native protein through immunoblotting, immunoprecipitation and immunofluorescence assays. Consistent with the presence of a glycosylphosphatidylinositol anchor, pypAg-2 fractionated with the detergent phase of Triton X-114-solubilized proteins and could be metabolically labeled with [3H]palmitic acid. By immunofluorescence, pypAg-2 expression was localized to both the trophozoite and merozoite membranes. Similar toPlasmodium falciparummerozoite surface protein 1, pypAg-2 contains two C-terminal epidermal growth factor (EGF)-like domains. Most importantly, immunization with recombinant pypAg-2 protected mice against lethalP. yoeliimalaria. Thus, pypAg-2 is a target of protective immune responses and represents a novel addition to the family of merozoite surface proteins that contain one or more EGF-like domains.
Augmentation of Nitric Oxide Production by Gamma Interferon in a Mouse Vascular Endothelial Cell Line and Its Modulation by Tumor Necrosis Factor Alpha and Lipopolysaccharide
Authors: Morikawa Akiko , Koide Naoki , Kato Yutaka , Sugiyama Tsuyoshi , Chakravortty Dipshikha , Yoshida Tomoaki , Yokochi Takashi
Published: July 2002

The effect of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide (LPS) on nitric oxide (NO) production in the mouse vascular aortic endothelial cell line END-D was examined. LPS, TNF-α, and a low concentration of IFN-γ inhibited NO production in END-D cells, while a high concentration of IFN-γ definitely enhanced it. The NO production induced by a high concentration of IFN-γ was further augmented by using IFN-γ in combination with LPS or TNF-α. In sequential incubations of LPS and IFN-γ, the enhancement of NO production required prior treatment with IFN-γ. Stimulation of END-D cells with a high concentration of IFN-γ led to the expression of inducible NO synthase (iNOS). The augmentation of NO production by IFN-γ alone or in combination with LPS or TNF-α was completely blocked by several inhibitors of iNOS. It was strongly suggested that a high concentration of IFN-γ itself enhanced NO production in END-D cells through inducing the expression of iNOS. LPS and TNF-α exclusively modulated the activity of iNOS once its expression was triggered by IFN-γ. On the other hand, a low concentration of IFN-γ, LPS, and TNF-α reduced NO production through down-regulating constitutive NOS (cNOS). The differential regulation of cNOS- and iNOS-mediated NO production by IFN-γ, TNF-α, and LPS is discussed.
Lack of CD4+T Cells Does Not Affect Induction of CD8+T-Cell Immunity againstEncephalitozoon cuniculiInfection
Authors: Moretto Magali , Casciotti Lori , Durell Brigit , Khan Imtiaz A.
Published: July 2002

Cell-mediated immunity has been reported to play an important role in defense againstEncephalitozoon cuniculiinfection. Previous studies from our laboratory have underlined the importance of cytotoxic CD8+T lymphocytes (CTL) in survival of mice infected withE. cuniculi. In the present study, immune response againstE. cuniculiinfection in CD4+T-cell-deficient mice was evaluated. Similar to resistant wild-type animals, CD4−/−mice were able to resolveE. cuniculiinfection even at a very high challenge dose (5 × 107spores/mouse). Tissues from infected CD4−/−mice did not exhibit higher parasite loads in comparison to the parental wild-type mice. Conversely, at day 21 postinfection, susceptible CD8−/−mice had 1014times more parasites in the liver compared to control wild-type mice. Induction of the CD8+T-cell response in CD4−/−mice againstE. cuniculiinfection was studied. Interestingly, a normal antigen-specific CD8+T-cell response toE. cuniculiinfection was observed in CD4−/−mice (precursor proliferation frequency, 1/2.5 × 104versus 1/104in wild-type controls). Lack of CD4+T cells did not alter the magnitude of the antigen-specific CTL response (precursor CTL frequency; 1/1.4 × 104in CD4−/−mice versus 1/3 × 104in control mice). Adoptive transfer of immune CD8+T cells from both CD4−/−and wild-type animals prevented the mortality in CD8−/−mice.E. cuniculiinfection thus offers an example of an intracellular parasitic infection where CD8+T-cell immunity can be induced in the absence of CD4+T cells.
Suppression of Gamma Interferon Transcription and Production by Nematode Excretory-Secretory Antigen during Polyclonal Stimulation of Rat Lymph Node T Cells
Authors: Uchikawa Ryuichi , Matsuda Shinji , Arizono Naoki
Published: July 2002

Although certain helminth infections preferentially induce type 2 T-cell responses, the immunological mechanisms responsible for type 2 T-cell polarization remain unclear. In the present study, we investigated the effects of excretory-secretory (ES) antigen from the nematodeNippostrongylus brasiliensison cytokine production by mesenteric lymph node (MLN) cells isolated from naive rats. MLN cells produced considerable levels of gamma interferon (IFN-γ) during a 72-h stimulation with concanavalin A (ConA) or with immobilized anti-CD3 plus soluble anti-CD28 antibodies (anti-CD3/CD28). With either stimulation, 10 μg of ES antigen per ml significantly suppressed IFN-γ and interleukin-2 (IL-2) production without cytotoxic activity. The copresence of anti-IL-4, anti-IL-10, or transforming growth factor β (TGF-β) blocking antibodies did not alter the suppressive effect of ES antigen on IFN-γ production. ES antigen did not affect IL-10 production. Kinetic studies of the effect of ES antigen indicated that the antigen suppressed even ongoing IFN-γ production. Reverse transcription-PCR study showed that in the presence of ES antigen, IFN-γ mRNA expression by MLN cells was suppressed 6 and 12 h after ConA or anti-CD3/CD28 stimulation. ES antigen also significantly suppressed IFN-γ production by purified CD4+or CD8+T cells during anti-CD3/CD28 stimulation but did not affect IL-4 production by CD4+T cells. These findings suggested that the nematode antigen suppressed production of IFN-γ and IL-2 but not IL-4 or IL-10 production. ES antigen-mediated suppression of IFN-γ during the initiation of the immune response may provide a microenvironment that helps generation of type 2 T cells.
Human Neutrophil-Mediated Nonoxidative Antifungal Activity againstCryptococcus neoformans
Authors: Mambula Salamatu S. , Simons Elizabeth R. , Hastey Ryan , Selsted Michael E. , Levitz Stuart M.
Published: July 2002

It has long been appreciated that polymorphonuclear leukocytes (PMN) killCryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing ofC. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation ofC. neoformanswith these fractions for 18 h resulted in percents inhibition of growth of 67.4 ± 3.4, 84.6 ± 4.4, and 29.2 ± 10.5 (mean ± standard error,n= 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C4column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.
Mononuclear Cell Recruitment, Granuloma Assembly, and Response to Treatment in Experimental Visceral Leishmaniasis: Intracellular Adhesion Molecule 1-Dependent and -Independent Regulation
Authors: Murray Henry W.
Published: July 2002

In experimental visceral leishmaniasis, acquired resistance to intracellularLeishmania donovaniis Th1 cell cytokine dependent and largely mediated by gamma interferon (IFN-γ); the same response also permits conventional antimony (Sb) chemotherapy to express its leishmanicidal effect. Since the influxing blood monocyte (which utilizes endothelial cell ICAM-1 for adhesion and tissue entry) is a primary effector target cell for this cytokine mechanism, we tested the monocyte's role in host responsiveness to chemotherapy in mice with ICAM-1 gene disruptions. Mutant animals failed to develop any early granulomatous tissue response in the liver, initially supported high-level visceral parasite replication, and showed no killing after Sb treatment; the leishmanicidal response to a directly acting, alternative chemotherapeutic probe, amphotericin B, was intact. However, mutant mice proceeded to express a compensatory, ICAM-1-independent response leading to mononuclear cell influx and granuloma assembly, control over visceral infection, and the capacity to respond to Sb. Together, these results point to the recruitment of emigrant monocytes and mononuclear cell granuloma formation, mediated by ICAM-1-dependent and -independent pathways, as critical determinants of host responsiveness to conventional antileishmanial chemotherapy.
Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells
Authors: Dziewanowska Katarzyna , Carson Andrew R. , Patti Joseph M. , Deobald Claudia F. , Bayles Kenneth W. , Bohach Gregory A.
Published: July 2002

We reported previously that internalization of
Staphylococcus aureus
by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673–4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of
S. aureus
by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for β
1
integrins dramatically reduced
S. aureus
invasion, suggesting that the formation of a Fn bridge linking the host cell β
1
integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional ∼55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of
S. aureus
. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to β
1
integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, β
1
integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the
Neisseria gonorrhoeae
FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369–379, 1998).
Role of Catalase in
Campylobacter jejuni
Intracellular Survival
Authors: Day William A. , Sajecki Jaime L. , Pitts Todd M. , Joens Lynn A.
Published: July 2002

The ability of
Campylobacter jejuni
to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of
C. jejuni
intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic
sod
mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to
C. jejuni
intracellular survival. Zymographic analysis indicated that
C. jejuni
expresses a single catalase enzyme. The gene encoding catalase (
katA
) was cloned via functional complementation, and an isogenic
katA
mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of
C. jejuni
growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and
katA
mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type
C. jejuni
; in contrast, no viable
katA
mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of
katA
mutant
C. jejuni
. These observations indicate that catalase is essential for
C. jejuni
intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.
Essential Role of Platelet-Activating Factor in Control ofLeishmania(Leishmania)amazonensisInfection
Authors: Lonardoni Maria Valdrinez Campana , Russo Momtchillo , Jancar Sonia
Published: July 2002

In the present study we investigated the role of platelet-activating factor (PAF) and prostaglandins in experimentalLeishmania(Leishmania)amazonensisinfection and the relationship between these mediators and nitric oxide (NO) production. Mouse peritoneal macrophages elicited with thioglicolate were infected with leishmania amastigotes, and the infection index determined 48 h later. The course of infection was monitored for 5 weeks in mice infected in the footpad with promastigotes by measuring the footpad swelling and parasite load in regional lymph nodes and spleen. The addition of PAF to C57BL/6 mouse macrophages significantly inhibited parasite growth and induced NO production. Treatment of macrophages with a selective PAF antagonist, WEB2086, increased the infection, indicating that endogenously produced PAF regulates macrophage ability to control leishmania infection. This effect of PAF was abolished by addition of the inhibitor of NO synthesis, L-NAME, to the cultures. The addition of prostaglandin E2significantly increased the infection and NO production. Treatment with cyclo-oxygenase inhibitor, indomethacin, reduced the infection and PAF-induced release of NO. Thus, the increased NO production induced by PAF seems to be mediated by prostaglandins. The more-selective inhibitors of cyclo-oxygenase 2, nimesulide and NS-398, had no significant effect. Thus, antileishmanial activity correlates better with the presence of PAF or absence of prostaglandins than with NO production. In vivo treatment with PAF antagonists significantly increased leishmania lesions, as well as the parasite load, in regional lymph nodes and spleens. These findings indicate that PAF is essential for the control of leishmania infection.
Yersinia pestis
YscG Protein Is a Syc-Like Chaperone That Directly Binds YscE
Authors: Day James B. , Guller Inna , Plano Gregory V.
Published: July 2002

Pathogenic
Yersinia
species secrete virulence proteins, termed
Yersinia
outer proteins (Yops), upon contact with a eukaryotic cell. The secretion machinery is composed of 21
Yersinia
secretion (Ysc) proteins.
Yersinia pestis
mutants defective in expression of YscG or YscE were unable to export the Yops. YscG showed structural and limited amino-acid-sequence similarities to members of the specific Yop chaperone (Syc) family of proteins. YscG specifically recognized and bound YscE; however, unlike previously characterized Syc substrates, YscE was not exported from the cell. These data suggest that YscG functions as a chaperone for YscE.
Association of Protease Activity in
Vibrio cholerae
Vaccine Strains with Decreases in Transcellular Epithelial Resistance of Polarized T84 Intestinal Epithelial Cells
Authors: Mel Stephanie F. , Fullner Karla Jean , Wimer-Mackin Susan , Lencer Wayne I. , Mekalanos John J.
Published: July 2002

Culture supernatants prepared from reactogenic strains of
Vibrio cholerae
cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.
Inhibition of Leukocyte Entry into the Brain by the Selectin Blocker Fucoidin Decreases Interleukin-1 (IL-1) Levels but Increases IL-8 Levels in Cerebrospinal Fluid during Experimental Pneumococcal Meningitis in Rabbits
Authors: Østergaard Christian , Yieng-Kow Runa Vavia , Benfield Thomas , Frimodt-Møller Niels , Espersen Frank , Lundgren Jens D.
Published: July 2002

The polysaccharide fucoidin is a selectin blocker that inhibits leukocyte recruitment into the cerebrospinal fluid (CSF) during experimental pneumococcal meningitis. In the present study, the effect of fucoidin treatment on the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-8 into the CSF was investigated. Rabbits (
n
= 7) were treated intravenously with 10 mg of fucoidin/kg of body weight every second hour starting 4 h after intracisternal inoculation of ∼10
6
CFU of
Streptococcus pneumoniae
type 3 (untreated control group,
n
= 7). CSF samples were obtained every second hour during a 16-h study period. Treatment with fucoidin caused a consistent and significant decrease in CSF IL-1 levels (in picograms per milliliter) between 12 and 16 h (0 versus 170, 0 versus 526, and 60 versus 1,467, respectively;
P
< 0.02). A less consistent decrease in CSF TNF-α levels was observed in the fucoidin-treated group, but with no significant difference between the two groups (
P
> 0.05). In contrast, there was no attenuation in CSF IL-8 levels. Indeed, there was a significant increase in CSF IL-8 levels (in picograms per milliliter) in the fucoidin-treated group at 10 and 12 h (921 versus 574 and 1,397 versus 569, respectively;
P
< 0.09). In conclusion, our results suggest that blood-derived leukocytes mainly are responsible for the release of IL-1 and to some degree TNF-α into the CSF during pneumococcal meningitis, whereas IL-8 may be produced by local cells within the brain.
Type III Secretion-Dependent Cell Cycle Block Caused in HeLa Cells by Enteropathogenic
Escherichia coli
O103
Authors: Nougayrède Jean-Philippe , Boury Michèle , Tasca Christian , Marchès Olivier , Milon Alain , Oswald Eric , De Rycke Jean
Published: July 2002

Rabbit enteropathogenic
Escherichia coli
(EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13

suc1

-agarose beads. This shows that Cdk1 is implicated in the G
2
/M arrest caused by EPEC strain E22.
Adoptive Transfer of CD4+T Cells Specific for Subunit A ofHelicobacter pyloriUrease ReducesH. pyloriStomach Colonization in Mice in the Absence of Interleukin-4 (IL-4)/IL-13 Receptor Signaling
Authors: Lucas Bernadette , Bumann Dirk , Walduck Anna , Koesling Jan , Develioglu Leyla , Meyer Thomas F. , Aebischer Toni
Published: July 2002

Protection in the murine model ofHelicobacter pyloriinfection may be mediated by CD4+T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterizedH. pyloriurease-specific CD4+T cells from BALB/c mice immunized withSalmonella entericaserovar Typhimurium expressingH. pyloriurease (subunits A and B). The CD4+T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-γ), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-γ and IL-10. Adoptive transfer of the UreA-specific CD4+T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+T cells into IL-4 receptor α chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis ofH. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.
Mycobacterium bovis BCG-Induced Granuloma Formation Depends on Gamma Interferon and CD40 Ligand but Does Not Require CD28
Authors: Hogan Laura H. , Markofski Wes , Bock Anja , Barger Brittany , Morrissey James D. , Sandor Matyas
Published: July 2002

Progressive granuloma formation is a hallmark of chronic mycobacterial infection. Granulomas are localized, protective inflammatory reactions initiated by CD4+T cells, which contribute to control of bacterial growth and blockade of bacterial dissemination. In order to understand the costimulatory requirements that allow CD4+T cells to directly or indirectly induce granulomas, we studied granuloma formation after 6 weeks inMycobacterium bovisBCG-infected CD28- and CD40 ligand (CD40L)-deficient mice and compared it to granuloma formation in infected wild-type inbred mice and infected cytokine-deficient mice. We characterized granulomas morphologically in liver sections, analyzed granuloma infiltrating cells by flow cytometry, and measured cytokine production by cultured granuloma cells. CD28-deficient mice have no defect at the local inflammatory site, inasmuch as they form protective granulomas and control bacterial growth. However, there are fewer activated T cells in the spleen compared to infected wild-type animals, and quantitative differences in the cellular composition of the granuloma are observed by flow cytometry. In CD40L-deficient mice, the granuloma phenotype is very similar to the phenotype in gamma interferon (IFN-γ)-deficient mice. Both IFN-γ-deficient and CD40L-deficient mice form granulomas which prevent bacterial dissemination, but control of bacterial growth is significantly impaired. The relative proportion of CD4+T cells in granulomas from both CD28−/−and CD40L−/−mice is significantly decreased compared with wild-type animals. Both models demonstrate that the phenotype and activation stage of systemic T cells do not always correlate with the phenotype and activation stage of the localized granulomatous response.
Complete DNA Sequence and Comparative Analysis of the 50-Kilobase Virulence Plasmid of
Salmonella enterica
Serovar Choleraesuis
Authors: Haneda Takeshi , Okada Nobuhiko , Nakazawa Noriko , Kawakami Takatoshi , Danbara Hirofumi
Published: July 2002

The complete nucleotide sequence of pKDSC50, a large virulence plasmid from
Salmonella enterica
serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the
spv
operon, which is composed of genes essential for systemic infection by nontyphoidal
Salmonella
. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the
spvRABCD
genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the
pef
(plasmid-encoded fimbria) operon and
rck
(resistance to complement killing) gene, which has been identified on the virulence plasmid of
S. enterica
serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic
Escherichia coli
O157:H7 and the enteropathogenic
E. coli
(EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of
Salmonella
serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.
Conformational Nature of the
Borrelia burgdorferi
Decorin Binding Protein A Epitopes That Elicit Protective Antibodies
Authors: Ulbrandt Nancy D. , Cassatt David R. , Patel Nita K. , Roberts William C. , Bachy Christine M. , Fazenbaker Christine A. , Hanson Mark S.
Published: July 2002

Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental
Borrelia burgdorferi
infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from
B. burgdorferi
strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous
B. garinii
and
B. afzelii
strains. Additionally, nonsecreted recombinant forms of DbpA
N40
were found to be inferior to secreted lipoprotein DbpA
N40
in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.
Mutational Analysis of the
tra
Locus of the Broad-Host-Range
Streptomyces
Plasmid pIJ101
Authors: Pettis Gregg S. , Cohen Stanley N.
Published: July 2002

The
tra
gene of
Streptomyces lividans
plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the
tra
gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211–6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.
Heterologous NNR-Mediated Nitric Oxide Signaling in
Escherichia coli
Authors: Hutchings Matthew I. , Shearer Neil , Wastell Sarah , van Spanning Rob J. M. , Spiro Stephen
Published: July 2002

The transcription factor NNR from
Paracoccus denitrificans
was expressed in a strain of
Escherichia coli
carrying a plasmid-borne fusion of the
melR
promoter to
lacZ
, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO
+
donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the
E. coli
reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a “positive control” phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
Purification and Characterization of an Iron Superoxide Dismutase and a Catalase from the Sulfate-Reducing Bacterium
Desulfovibrio gigas
Authors: Dos Santos Wagner G. , Pacheco Isabel , Liu Ming-Yih , Teixeira Miguel , Xavier António V. , LeGall Jean
Published: July 2002

The iron-containing superoxide dismutase (FeSOD; EC
1.15.1.1
) and catalase (EC
1.11.1.6
) enzymes constitutively expressed by the strictly anaerobic bacterium
Desulfovibrio gigas
were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms,
D. gigas
FeSOD is sensitive to H
2
O
2
and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand,
D. gigas
catalase has an estimated molecular mass of 186 ± 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H
2
O
2
and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (
g

y
,
x

= 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.
Towards Single-Copy Gene Expression Systems Making Gene Cloning Physiologically Relevant: Lambda InCh, a Simple
Escherichia coli
Plasmid-Chromosome Shuttle System
Authors: Boyd Dana , Weiss David S. , Chen Joseph C. , Beckwith Jon
Published: July 2002

We describe a simple system for reversible, stable integration of plasmid-borne genes into the
Escherichia coli
chromosome. Most ordinary
E. coli
strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used. A single genetic element, a lambda phage, is the only specialized vector required. The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted.