bluuebunny/crossref_metadata_2025 huggingface dataset:
Capsular Polysaccharide and O-Specific Antigen Divergently Modulate Pulmonary Neutrophil Influx in an
Escherichia coli
Model of Gram-Negative Pneumonitis in Rats
Authors: Russo Thomas A. , Davidson Bruce A. , Priore Roger L. , Carlino Ulrike B. , Helinski Jadwiga D. , Knight Paul R
Published: July 2002

Enteric gram-negative bacilli cause a severe, often life-threatening pneumonia. An improved understanding of the pathogenesis of this infection may lead to improved treatment. Nearly all of the responsible gram-negative bacilli possess capsular polysaccharides and/or an O-specific antigen as part of their lipopolysaccharide (LPS). We hypothesized that these surface polysaccharides may modulate the pulmonary host response. To investigate this, a rat pneumonitis model was used, and pulmonary neutrophil influx, a critical aspect of host defense, was measured. To assess for the effect of the capsule and O-specific antigen on this host response, three proven, isogenic derivatives that are deficient in capsular polysaccharide alone (CP9.137), the O-specific antigen moiety of the LPS alone (CP921), and both the capsular polysaccharide and O-specific antigen (CP923), as well as their wild-type parent (CP9), were used as challenge strains at various intratracheal challenge inocula (CI). Total lung myeloperoxidase (MPO), a surrogate marker for neutrophils, was measured for 15 h post-bacterial challenge. To determine the effect of capsule and the O-specific antigen on the measured MPO levels, a mathematical model was developed and used to describe the MPO levels as a function of time for each CI of each of the four strains. The results from this analysis demonstrated that in the absence of the K54 capsule, 80.7 times the CI is necessary to achieve the same maximum MPO level relative to K54 positive strains (
P
< 0.0001). In contrast, a diametric effect was observed in the absence of the O-specific antigen, where 0.13 times the CI was necessary to achieve the same maximum MPO level relative to O4-positive strains (
P
= 0.0032). No interactive effect was observed between the capsule and the O-specific antigen. These findings demonstrate that these surface polysaccharides modulate pulmonary neutrophil influx and suggest that the K54 capsular polysaccharide is a proinflammatory mediator and that the O4-specific antigen attenuates the proinflammatory response. If these speculations are substantiated, an understanding of how the capsule and the O-specific antigen modulate host response could have significant therapeutic implications. The potential use of biologic modulators directed against the host response, as well as approaches based on inactivating bacterial components (e.g., surface polysaccharides) in attempts to modify sepsis syndromes, could be developed.
A Commercial Preparation of Catalase Inhibits Nitric Oxide Production by Activated Murine Macrophages: Role of Arginase
Authors: Tian Y. , Xing Y. , Magliozzo R. , Yu K. , Bloom B. R. , Chan J.
Published: July 2002

Catalase is widely used as a pharmacological probe to evaluate the role of hydrogen peroxide in antimicrobial activities of phagocytic cells. This report demonstrates that the ability of a commercial preparation of catalase to inhibit concomitantly macrophage antimycobacterial activity and production of reactive nitrogen intermediates can be attributed, at least in part, to the depletion of
l
-arginine by contaminating arginase. In experimental systems that employ pharmacological probes, the existence of nonspecific effects should be considered in data interpretation.
Evidence That Hyaluronidase Is Not Involved in Tissue Invasion of the Protozoan Parasite
Entamoeba histolytica
Authors: Nickel Rosa , Stern Robert , Leippe Matthias
Published: July 2002

As previous reports suggested that a hyaluronidase is involved in tissue invasion of
Entamoeba histolytica
, we searched for such an activity in trophozoite extracts. A hyaluronidase activity was not detectable in long-term cultures or in amoebae freshly passaged through a gerbil liver, as evidenced by four different techniques.
Replacement of Histidine 340 with Alanine Inactivates the Group A
Streptococcus
Extracellular Cysteine Protease Virulence Factor
Authors: Gubba Siddeswar , Cipriano Vincent , Musser James M.
Published: July 2002

Streptococcus pyogenes
expresses a highly conserved extracellular cysteine protease that is a virulence factor for invasive disease, including soft tissue infection. Site-directed mutagenesis was used to generate a His340Ala recombinant mutant protein that was made as a stable 40-kDa zymogen by
Escherichia coli
. Purified His340Ala protein was proteolytically inactive when bovine casein and human fibronectin were used as substrates. Wild-type 28-kDa streptococcal protease purified from
S. pyogenes
processed the 40-kDa mutant zymogen to a 28-kDa mature form, a result suggesting that the derivative protein retained structural integrity. The data are consistent with the hypothesis that His340 is an enzyme active site residue, an idea confirmed by recent solution of the zymogen crystal structure (T. F. Kagawa, J. C. Cooney, H. M. Baker, S. McSweeney, M. Liu, S. Gubba, J. M. Musser, and E. N. Baker, Proc. Natl. Acad. Sci. USA 97:2235–2240, 2000). The data provide additional insight into structure-function relationships in this
S. pyogenes
virulence factor.
Lipoarabinomannans Activate the Protein Tyrosine Kinase Hck in Human Neutrophils
Authors: Astarie-Dequeker Catherine , Nigou Jérôme , Puzo Germain , Maridonneau-Parini Isabelle
Published: July 2002

The mycobacterial lipoarabinomannans (LAMs) are glycosylphosphatidyl-myo-inositol-anchored lipoglycans with diverse biological activities. It has been shown that purified LAMs interact directly, or indirectly, through receptors with the plasma membrane receptors of target cells located in domains rich in glycosylphosphatidylinositol-anchored proteins that contain Src family protein tyrosine kinases. To examine whether LAMs could activate Src-related kinases, human neutrophils were exposed to mannosylated LAMs (ManLAMs) purified from the vaccinal strainMycobacterium bovisBCG and to phosphoinositol-capped LAMs (AraLAM or PILAM) obtained from the nonpathogenic speciesMycobacterium smegmatis. We report first that both ManLAMs and PILAMs activate Hck in a rapid and transient manner and second that complete deacylation of ManLAM abolished its effect on Hck activity, thereby demonstrating that acylation of LAM but not mannosylation is critical for Hck activation. These data indicate that Hck is involved in the signaling pathway of LAMs, molecules known for their ability to trigger several responses in eukaryotic cells.
Extract ofNippostrongylus brasiliensisStimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch
Authors: Ehigiator Humphrey N. , Stadnyk Andrew W. , Lee Timothy D. G.
Published: July 2002

Infection with the nematode parasiteNippostrongylus brasiliensisinduces a pronounced type-2 T-cell response that is associated with marked polyclonal immunoglobulin E (IgE) and IgG1 production in mice. To examine the differential roles of the infection and products produced by nematodes, we investigated a soluble extract ofN. brasiliensisfor the ability to mediate this type-2 response. We found that the extract induced a marked increase in IgE and IgG1 levels, similar to that induced by the infection. The extract did not affect the level of IgG2a in serum, showing that the effect was specific to IgE and IgG1 (type-2-associated immunoglobulin) rather than inducing a nonspecific increase in all immunoglobulin isotypes. This response was also associated with increased interleukin-4 production in vitro. These results confirm that the extract, like infection, is a strong inducer of polyclonal type-2 responses and a reliable model for investigating the regulation of nematode-induced responses. The extract induced the production of IgG1 when added to in vitro cultures of lipopolysaccharide-stimulated B cells. This provides evidence for the induction of class switch. It did not induce upregulation of IgG1 in naive (unstimulated) B cells or expand B cells in in vitro cultures. Analysis of DNA from the spleens of mice treated with the extract by digestion-circularization PCR demonstrated a marked increase in the occurrence of γ1 switch region gene recombination in the cells in vivo. These results provide strong evidence that soluble worm products are able to mediate the marked polyclonal γ1/ɛ response and that infection is not required to mediate this response. Furthermore, these data provide evidence that the soluble nematode extract induces this effect by causing de novo class switch of B cells and not by an expansion of IgG1 B cells or an increase in antibody production by IgG1 plasma cells.
Molecular Cloning, Sequencing, Expression, and Characterization of an Immunogenic 43-Kilodalton Lipoprotein of
Bartonella bacilliformis
That Has Homology to NlpD/LppB
Authors: Padmalayam Indira , Kelly Timothy , Baumstark Barbara , Massung Robert
Published: July 2002

A recombinant clone expressing an immunoreactive antigen of
Bartonella bacilliformis
was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein. Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen of
B. bacilliformis
is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins. This is the first report to date that characterizes a lipoprotein of
B. bacilliformis
. The immunogenicity of the
B. bacilliformis
LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis. Sera from patients who had a high titer for
Bartonella henselae
, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in
B. henselae
. Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of
B. bacilliformis
was generated. This antiserum did not recognize the NlpD homologue of
Escherichia coli
or the 43-kDa antigen of
B. henselae
.
T-Cell Responses to Immunodominant LACK Antigen Do Not Play a Critical Role in Determining Susceptibility of BALB/c Mice toLeishmania mexicana
Authors: Torrentera Fabiola Aguilar , Glaichenhaus Nicolas , Laman Jon D. , Carlier Yves
Published: July 2002

Although BALB/c mice develop lesions when infected withLeishmania mexicana, the mechanisms which are responsible for susceptibility to this parasite have not been elucidated. In contrast, susceptibility of BALB/c mice toLeishmania majorhas been shown to depend on the early production of interleukin-4 (IL-4) by T cells which react to the parasitic LACK antigen. Here, we demonstrate that the lesions induced byL. mexicanaare delayed compared to those induced byL. majorbut rapidly develop at later time points. Interestingly, while LACK-tolerant BALB/c-derived IE-LACK transgenic mice were resistant toL. major, they were susceptible toL. mexicanaand developed lesions similar to those observed in wild-type BALB/c mice. The latter result was observed despite the fact that (i) LACK was expressed byL. mexicana, (ii) splenocytes from BALB/c mice were able to stimulate LACK-specific T-cell hybridoma cells when incubated with liveL. mexicanapromastigotes, and (iii) LACK-specific T cells contributed to IL-4 production inL. mexicana-infected BALB/c mice. Thus, in contrast to what was observed forL. major-infected mice, LACK-specific T cells do not play a critical role in determining susceptibility toL. mexicana. Although BALB/c mice are susceptible to bothL. majorandL. mexicana, the mechanisms which are responsible for susceptibility to these parasites are likely to be different.
Soluble Pilin of
Neisseria gonorrhoeae
Interacts with Human Target Cells and Tissue
Authors: Rytkönen Anne , Johansson Linda , Asp Vendela , Albiger Barbara , Jonsson Ann-Beth
Published: July 2002

Pili of
Neisseria gonorrhoeae
are phase-variable surface structures that mediate adherence to host target cells. Each pilus is composed of thousands of major pilus subunits, pilins, pilus-associated protein PilC, and possibly other components. Piliated and nonpiliated gonococcal clones may secrete a soluble smaller pilin (S-pilin) that is cleaved after amino acid 39 of the mature pilin protein. Here, purified S-pilin was found to migrate as a 61- to 64-kDa double band on nondenaturing gels, suggesting the formation of tetrameric S-pilin proteins with two isomeric forms. In situ studies of binding to formalin-fixed tissue sections demonstrated the binding of S-pilin to human tissue but not to tissue from mouse or rat organs, showing the presence of a human-specific receptor-binding domain within the pilin polypeptide. Pretreatment of the target tissues with proteinase K decreased gonococcal binding dramatically, whereas pretreatment with neuraminidase and
meta
-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect gonococcal binding. In overlay assays, purified S-pilin bound to a band with a migration pattern and size similar to those of CD46, a cellular pilus receptor. Further, binding of
N. gonorrhoeae
to target cells and tissues could be blocked by both CD46 antibodies and purified S-pilin. These data argue that S-pilin interacts with a protein domain(s) of the CD46 receptor on human cells.
Parameters Underlying Successful Protection with Live Attenuated Mutants in Experimental Shigellosis
Authors: Bernardini Maria Lina , Arondel Josette , Martini Irene , Aidara Awa , Sansonetti Philippe J.
Published: July 2002

Because the use of live attenuated mutants of
Shigella
spp. represents a promising approach to protection against bacillary dysentery (M. E. Etherridge, A. T. M. Shamsul Hoque, and D. A. Sack, Lab. Anim. Sci. 46:61–66, 1996), it becomes essential to rationalize this approach in animal models in order to optimize attenuation of virulence in the vaccine candidates, as well as their route and mode of administration, and to define the correlates of protection. In this study, we have compared three strains of
Shigella flexneri
5—the wild-type M90T, an
aroC
mutant, and a double
purE aroC
mutant—for their pathogenicity, immunogenicity, and protective capacity. Protection against keratoconjunctivitis, induced by wild-type M90T, was used as the protection read out in guinea pigs that were inoculated either intranasally or intragastrically. Following intranasal immunization, the
aroC
mutant elicited weak nasal tissue destruction compared to M90T and achieved protection correlated with high levels of local anti-lipopolysaccharide immunoglobulin A (IgA), whereas the
purE aroC
double mutant, which also elicited weak tissue destruction, was not protective and elicited a low IgA response. Conversely, following intragastric immunization, only the M90T
purE aroC
double mutant elicited protection compared to both the
aroC
mutant and the wild-type strain. This mutant caused mild inflammatory destruction, particularly at the level of Peyer's patches, but it persisted much longer within the tissues. This could represent an essential parameter of the protective response that, in this case, did not clearly correlate with high anti-lipopolysaccharide IgA titers.
Expression, Extracellular Secretion, and Immunogenicity of thePlasmodium falciparumSporozoite Surface Protein 2 inSalmonellaVaccine Strains
Authors: Gómez-Duarte Oscar G. , Pasetti Marcela F. , Santiago Araceli , Sztein Marcelo B. , Hoffman Stephen L. , Levine Myron M.
Published: July 2002

Deleting transmembrane α-helix motifs fromPlasmodium falciparumsporozoite surface protein (SSP-2) allowed its secretion fromSalmonella entericaserovar Typhimurium SL3261 andS. entericaserovar Typhi CVD 908-htrAby the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P= 0.04).
Murine Model for Lymphocytic Tropism by
Borrelia burgdorferi
Authors: Dorward David W. , Larson Ralph S.
Published: July 2002

In vitro studies have demonstrated direct interactions between
Borrelia burgdorferi
and human B and T cells. However, largely because disseminated infections typically occur at very low density, little is known about associations between spirochetes and mammalian host cells in vivo. To assess whether spirochetes interact directly with lymphocytes in mammals, we developed a mouse model for lymphotropism. By repeatedly coincubating spirochetes with primary mouse lymphocytes that were immobilized by adherence to immunomagnetic beads, we were able to preferentially enrich cultures for or against bacteria with constitutive affinity for murine B and T cells. Populations of lymphotropically enriched, stock infectious, and lymphotropically depleted spirochetes were injected intradermally into mice. Lymphocytes were then purified from the blood and spleens of challenged mice and placed into spirochetal culture medium. Cultures of
B. burgdorferi
were obtained from primary lymphocyte preparations from mice challenged with each of the three populations of spirochetes. Recovery of lymphocyte-associated bacteria occurred within 1 h of challenge with enriched bacteria. Lymphocyte preparations from mice challenged with stock infectious and lymphotropically depleted bacteria produced cultures after 1 day postchallenge. All lymphocyte preparations were culture negative after 1 week. These results demonstrate that lymphotropic
B. burgdorferi
is infectious in mice and suggest that associations between spirochetes and lymphocytes occur in vivo. The results also suggest that factors involved in lymphocytic binding may be inducible in vivo. Thus, this system provides a model for studying the role of such interactions in mammalian infections.
Sequence and Antigenic Variability of the
Helicobacter mustelae
Surface Ring Protein Hsr
Authors: Forester Natasha , Lumsden John S. , O'Croinin Tadhg , O'Toole Paul W.
Published: July 2002

We have identified an array of more than 500 repetitive sequences flanking the
hsr
gene, which encodes the major surface protein of the ferret pathogen
Helicobacter mustelae
. The repeats show identity exclusively to the amino-terminal half of Hsr. Analysis of Hsr from three strains indicated variability of exposed epitopes. Characterization of an
hsr
mutant showed that Hsr is not an adhesin.
Chlamydia trachomatis
Infection Does Not Enhance Local Cellular Immunity against Concurrent
Candida
Vaginal Infection
Authors: Kelly Kathleen A. , Gray Heather L. , Walker Jennifer C. , Rank Roger G. , Wormley Floyd L. , Fidel Paul L.
Published: July 2002

Although Th1-type cell-mediated immunity (CMI) is the predominant host defense mechanism against mucosal
Candida albicans
infection, CMI against a vaginal
C. albicans
infection in mice is limited at the vaginal mucosa despite a strong
Candida
-specific Th1-type response in the draining lymph nodes. In contrast, Th1-type CMI is highly effective against an experimental
Chlamydia trachomatis
genital tract infection. This study demonstrated through two independent designs that a concurrent
Candida
and
Chlamydia
infection could not accelerate or modulate the anti-
Candida
CMI response. Together, these results suggest that host responses to these genital tract infections are independent and not influenced by the presence of the other.
Trichinella spiralis
-Infected Muscle Cells: Abundant RNA Polymerase II in Nuclear Speckle Domains Colocalizes with Nuclear Antigens
Authors: Yao Chaoqun , Jasmer Douglas P.
Published: July 2002

Infection of mammalian skeletal muscle cells by
Trichinella spiralis
causes host nuclei to become polyploid (ca. 4N) and abnormally enlarged. It has been postulated that this enlargement reflects an infection-induced elevation of host transcription. Anthelmintic treatment of
T. spiralis
-infected rodents with mebendazole (MBZ) causes a reduction in the size of infected cell nuclei and a significant reduction in the total RNA content of individual infected muscle cells. A monoclonal antibody to the large subunit of RNA polymerase II (Pol II) was used here to assess the effects of infection on Pol II levels in isolated infected cell nuclei. Pol II was localized to speckle domains in isolated infected cell nuclei. Similar domains have been previously localized to sites of RNA synthesis or processing. When compared to the levels in nuclei from other, uninfected host cells, speckle-localized Pol II (SL-Pol II) levels were significantly elevated in infected cell nuclei by a mean of 3.9- to 6.8-fold. Nuclear antigens (NA) recognized by antibodies against
T. spiralis
localized to infected cell nuclei. By use of confocal microscopy, a subpopulation of NA was found colocalized with most speckle domains defined by Pol II. MBZ treatment of chronically infected mice, which depletes NA from infected cell nuclei, caused a significant depletion of SL-Pol II from infected cell nuclei. Control nuclei had a mean of 70% more SL-Pol II than MBZ-treated nuclei. The mean residual level of Pol II in these polyploid nuclei remained elevated by 120% over the level in 2N control nuclei. These observations may indicate two distinct effects of infection on Pol II levels in host cells.
Protection against Pulmonary Infection withPseudomonas aeruginosafollowing Immunization withP. aeruginosa-Pulsed Dendritic Cells
Authors: Worgall Stefan , Kikuchi Toshiaki , Singh Ravi , Martushova Katherine , Lande Leah , Crystal Ronald G.
Published: July 2002

To develop aPseudomonas aeruginosavaccine that allows the host immune system to select the antigens, we hypothesized that dendritic cells (DC) pulsed withP. aeruginosawould induce protective immunity against pulmonary infections withP. aeruginosa. Incubation of murine bone marrow-derived DC withP. aeruginosain vitro led to uptake ofP. aeruginosaand activation of the DC. Spleen-derived CD4+cells from mice immunized withP. aeruginosa-pulsed DC showed increased proliferation, demonstrating that DC pulsed withP. aeruginosawere capable of eliciting aP. aeruginosa-specific immune response. To evaluate ifP. aeruginosa-pulsed DC can induce protective immunity againstP. aeruginosapulmonary infection, DC incubated withP. aeruginosain vitro were administered systemically to syngeneic mice, and the mice were then challenged by intrapulmonary infection withP. aeruginosa(5 × 104CFU/mouse) 13 days later. Unimmunized control mice and mice who had previously received naive DC or DC stimulated with lipopolysaccharide orEscherichia colidied within 72 h. In contrast, 45% of mice receivingP. aeruginosa-pulsed DC demonstrated prolonged survival (>14 days). Finally, DC-pulsed with heat-inactivatedP. aeruginosaprotected CD8−/−but not CD4−/−mice, demonstrating that CD4+T cells were required for the DC pulsed withP. aeruginosato induce protective immunity.
Differential Roles of Interleukin-18 (IL-18) and IL-12 for Induction of Gamma Interferon by Staphylococcal Cell Wall Components and Superantigens
Authors: Stuyt Rogier J. L. , Netea Mihai G. , Kim Soo-Hyun , Novick Daniela , Rubinstein Menachem , Kullberg Bart-Jan , Van der Meer Jos W. M. , Dinarello Charles A.
Published: July 2002

The roles of endogenous cytokines induced by either intact staphylococcal microorganisms or staphylococcal exotoxins were examined using human whole-blood cultures. To accomplish this, interleukin-18 binding protein (IL-18BP) and tumor necrosis factor binding protein (TNFbp) were used to neutralize IL-18 and TNF, respectively, whereas an anti-IL-12 monoclonal antibody was used to neutralize IL-12 and the IL-1 receptor antagonist (IL-1Ra) was used to block IL-1 receptors. Heat-killedStaphylococcus epidermidisandStaphylococcus aureus, as well as the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcus enterotoxin B (SEB) induced gamma interferon (IFN-γ) production.Staphylococcusspp.-induced production of IFN-γ required the presence of endogenous IL-18, IL-12, and TNF. In contrast, TSST-1-induced IFN-γ was not significantly reduced in the presence of IL-18BP, anti-IL-12 antibodies, IL-1Ra, or anti-TNFbp. SEB-induced IFN-γ was significantly inhibited only by anti-IL-12 antibodies, indicating that endogenous IL-18, IL-1, and TNF are not required for SEB-induced IFN-γ. In conclusion, the mechanisms of IFN-γ stimulation by intact staphylococcal microorganisms and by exotoxins differ, and this is likely due to the different receptors which are triggered on the cell membranes. In contrast to its role in the interactions between staphylococci and host cells, IL-18 does not appear to play a major role in superantigen-induced IFN-γ.
Biological Characterization of Lipopolysaccharide fromTreponema pectinovorum
Authors: Kesavalu Lakshmyya , Falk Clinton W. , Davis Kenneth J. , Steffen Michelle J. , Xu Xiaoping , Holt Stanley C. , Ebersole Jeffrey L.
Published: July 2002

This study investigated the endotoxic and biological properties of purified lipopolysaccharide (LPS) isolated from an oral spirochete,Treponema pectinovorum. Endotoxicity, measured byLimulusamoebocyte lysate kinetic assay, showed that the LPS contained 1.28 endotoxin units per μg of purified LPS, which was approximately 4,000 times less thanEscherichia coliO55:B5 LPS. To determine in vivo endotoxicity, LPS responder mice were administered LPS following galactosamine (GalN) sensitization. The LPS induced neither endotoxic symptoms nor lethality for 96 h, suggesting negligible or very low endotoxicity. In contrast, infection with liveT. pectinovoruminduced 100% lethality within 12 h in GalN-sensitized LPS responder mice, indicating an endotoxin-like property of this treponeme. Heat-killed microorganisms exhibited no lethality in GalN-sensitized mice, suggesting that the endotoxicity was associated with heat-labile components. To determine cytokine and chemokine induction by LPS, human gingival fibroblasts were stimulated and secretion of interleukin 1β (IL-1β), granulocyte-macrophage colony-stimulating factor, gamma interferon, IL-6, IL-8, and monocyte chemoattractant protein 1 (MCP-1) was assessed. The purified LPS induced significant amounts of only IL-6, IL-8, and MCP-1, although they were substantially lower than levels after challenge with liveT. pectinovorum. After injection of LPS or live or heat-killedT. pectinovorum, serum was collected from mice and analyzed for proinflammatory cytokines IL-1β, tumor necrosis factor alpha (TNF-α), and IL-6. LPS induced only IL-6 consistently. Both live and heat-killedT. pectinovoruminduced serum IL-6, which was higher than the level detected following LPS administration. Importantly, live bacteria elicited systemic TNF-α and IL-1β levels similar to those induced by a lethal dose of liveE. coliO111. The results indicated thatT. pectinovorumLPS has very low or no endotoxicity, although it can elicit low levels of cytokines from host cells. In contrast to the LPS, liveT. pectinovorumdemonstrated in vivo toxicity, which was associated with serum IL-1β, TNF-α, and IL-6, suggesting an endotoxin-like property of a heat-labile molecule(s) of the spirochete.
RecombinantDirofilaria immitisPolyprotein That Stimulates Murine B Cells To Produce Nonspecific Polyclonal Immunoglobulin E Antibody
Authors: Tezuka Hiroyuki , Imai Shinjiro , Muto Riho , Furuhashi Yuko , Fujita Koichiro
Published: July 2002

Nonspecific immunoglobulin E (IgE) production is an event characteristically observed in parasitic helminth infections, but its mechanisms are still unclear. To define these mechanisms, we prepared a recombinantDirofilaria immitisprotein (rDiAg) and assessed its effect on nonspecific IgE production. rDiAg preferentially induced nonspecific IgE production, without eliciting specific IgE production, as well as a Th2-type cytokine profile (high interleukin-4 [IL-4] and IL-10 production but low gamma interferon production) in BALB/c mice. rDiAg significantly elicited the proliferative response of naive B cells. This response was not abolished by polymyxin B, an inhibitor of lipopolysaccharide (LPS), and rDiAg normally expanded splenic B cells from LPS nonresponder C3H/HeJ mice. Thus, the mitogenic effect of rDiAg was not due to LPS contamination. rDiAg also enhanced levels of CD23 expression on splenic B cells. Splenic B cells produced marked levels of IgE when cultured with the combination of rDiAg and IL-4 (rDiAg-IL-4), whereas peritoneal B cells produced negligible levels of IgE. rDiAg-IL-4-induced IgE production by splenic B cells was synergistically increased by coculture with peritoneal B cells. rDiAg-driven IL-10 secretion was higher in peritoneal B cells than in splenic B cells. IgE production by splenic B cells cocultured with peritoneal B cells was decreased to a level comparable to that by splenic B cells in the presence of a neutralizing anti-IL-10 monoclonal antibody. Collectively, these results suggest that rDiAg-induced polyclonal expansion and IgE class switching of splenic B cells contribute to nonspecific IgE production and that these responses are enhanced by peritoneal B-cell-derived IL-10.
Regulation of Proinflammatory Cytokines in Human Lung Epithelial Cells Infected withMycoplasma pneumoniae
Authors: Yang Jun , Hooper W. Craig , Phillips Donald J. , Talkington Deborah F.
Published: July 2002

Mycoplasma pneumoniaeis a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown thatM. pneumoniaecan induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate thatM. pneumoniaeinfection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1β mRNA also increased after infection and IL-1β protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found thatM. pneumoniaecytadherence is important for the induction of cytokines. On the other hand, whileM. pneumoniaeprotein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis ofM. pneumoniaeinfection and provide a better understanding ofM. pneumoniaepathology at the cellular level.
Overexpression of the RNA Polymerase Alpha Subunit Reduces Transcription of Bvg-Activated Virulence Genes in
Bordetella pertussis
Authors: Carbonetti Nicholas H. , Romashko Alla , Irish Teresa J.
Published: July 2002

Overexpression of the RNA polymerase alpha subunit in
Bordetella pertussis
reduces expression of the virulence factor pertussis toxin. Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha.
A pAD1-Encoded Small RNA Molecule, mD, Negatively Regulates
Enterococcus faecalis
Pheromone Response by Enhancing Transcription Termination
Authors: Tomita Haruyoshi , Clewell Don B.
Published: July 2002

pAD1 is a 60-kb hemolysin-bacteriocin plasmid in
Enterococcus faecalis
that encodes a conjugative mating response to a peptide sex pheromone, cAD1, secreted by plasmid-free bacteria. The pheromone response is regulated by two proteins: TraE1, which positively regulates all or most conjugative structural genes, and TraA, which negatively regulates
traE1
. TraA binds to pAD1 DNA at the
iad
(encoding the inhibitor peptide iAD1) promoter but is released upon binding to imported pheromone. This leads to enhanced transcription through two closely spaced downstream terminators (t1 and t2) into
traE1
. TraE1 is believed to then upregulate itself from a site located within t2; thus, a small amount of transcription through t1-t2 could lead to overall induction. It is important therefore that the t1-t2 terminators be tightly controlled to keep the response shut down in the absence of pheromone. A small (200-nucleotide) RNA molecule designated mD is encoded just upstream of t1 by a determinant (
traD
) oriented in the direction opposite to that of transcripts utilizing t1. mD is expressed at high levels in the uninduced state, but it decreases significantly upon induction. Here we present results of genetic studies relating to the activity of t1-t2 and show that mD strongly enhances transcriptional termination at t1. The mD activity is shown to influence transcription well downstream and can affect the determinant for aggregation substance
asa1
. The phenomenon is specific in that there is no effect of mD on the unrelated pheromone-responding plasmids pPD1 and pCF10.
Requirement for Homologous Recombination Functions for Expression of the
mutA
Mistranslator tRNA-Induced Mutator Phenotype in
Escherichia coli
Authors: Ren Li , Mamun Abu Amar M. Al , Humayun M. Zafri
Published: July 2002

Expression of the
Escherichia coli mutA
mutator phenotype requires
recA
,
recB
,
recC
,
ruvA
, and
ruvC
gene, but not
recD
,
recF
,
recO
, or
recR
genes. Thus, the
recBCD
-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase in
mutA
cells.
The
Yersinia enterocolitica
Phospholipase Gene
yplA
Is Part of the Flagellar Regulon
Authors: Schmiel Deborah H. , Young Glenn M. , Miller Virginia L.
Published: July 2002

Yersinia enterocolitica yplA
encodes a phospholipase required for virulence. Virulence genes are often regulated in response to environmental signals; therefore,
yplA
expression was examined using a
yplA
::
lacZY
transcriptional fusion. Maximal
yplA
expression occurred between pH 6.5 and pH 7.5 and was induced in the mid-logarithmic growth phase. Potential Fnr, cyclic AMP (cAMP)-cAMP receptor protein (Crp), and ς
F
regulatory sites were identified in the nucleotide sequence. Reduction of
yplA
expression by aeration, addition of glucose and sucrose, and application of high temperature and salt is consistent with Fnr-, cAMP-Crp-, and ς
F
-mediated regulation, respectively. Expression of
yplA
was reduced in
flhDC
and
fliA
null strains, indicating that
yplA
is part of the flagellar regulon.
The
KlGpa1
Gene Encodes a G-Protein α Subunit That Is a Positive Control Element in the Mating Pathway of the Budding Yeast
Kluyveromyces lactis
Authors: Saviñón-Tejeda Alma L. , Ongay-Larios Laura , Valdés-Rodrı́guez Julián , Coria Roberto
Published: July 2002

The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a
Kluyveromyces lactis
genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon. The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein α subunit from
Saccharomyces cerevisiae
involved in the mating pheromone response pathway. Nevertheless, unlike disruption of
Gpa1
from
S. cerevisiae
, disruption of
KlGpa1
rendered viable cells with a reduced capacity to mate. Expression of a plasmidic
KlGpa1
copy in a Δ
Klgpa1
mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway. Overexpression of the constitutive subunit KlGpa1p(K
364
) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of Δ
Klgpa1
mutant cells.
K. lactis
expresses a second Gα subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue Δ
Klgpa1
cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by KlGpa1p and glucose metabolism mediated by KlGpa2p. The Δ
Klgpa1 ΔKlgpa2
double mutant, although viable, showed the mating deficiency observed in the single Δ
Klgpa1
mutant.
Functional Complementation of Pyran Ring Formation in Actinorhodin Biosynthesis in
Streptomyces coelicolor
A3(2) by Ketoreductase Genes for Granaticin Biosynthesis
Authors: Ichinose Koji , Taguchi Takaaki , Bedford David J. , Ebizuka Yutaka , Hopwood David A.
Published: July 2002

A mutation in
act
VI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by
Streptomyces coelicolor
, was complemented by
gra
-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of
Streptomyces violaceoruber
Tü22. It is hypothesized that, while
gra
-ORF5 alone is a ketoreductase for C-9,
gra
-ORF6 gives the enzyme regiospecificity also for C-3.
The Iron- and Temperature-Regulated
cjrBC
Genes of
Shigella
and Enteroinvasive
Escherichia coli
Strains Code for Colicin Js Uptake
Authors: Šmajs David , Weinstock George M.
Published: July 2002

A cosmid library of DNA from colicin Js-sensitive enteroinvasive
Escherichia coli
(EIEC) strain O164 was made in colicin Js-resistant strain
E. coli
VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping
senB
. The open reading frames were designated
cjrABC
(for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn
7
insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of
cjrA
, suggesting regulation of the
cjr
operon by iron levels. CjrA protein was homologous to iron-regulated
Pseudomonas aeruginosa
protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from
Pseudomonas putida
; and CjrC was homologous to a putative outer membrane siderophore receptor from
Campylobacter jejuni.
Cloning experiments showed that the
cjrB
and
cjrC
genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the
E. coli
K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.
Dual Regulatory Control of a Particle Maturation Function of Bacteriophage P1
Authors: Lehnherr Hansjörg , Jensen Charlotte D. , Stenholm Anne R. , Dueholm Anita
Published: July 2002

A unique arrangement of promoter elements was found upstream of the bacteriophage P1 particle maturation gene (
mat
). A P1-specific late-promoter sequence with conserved elements located at positions −22 and −10 was expected from the function of the gene in phage morphogenesis. In addition to a late-promoter sequence, a −35 element and an operator sequence for the major repressor protein, C1, were found. The −35 and −10 elements constituted an active
Escherichia coli
ς
70
consensus promoter, which was converted into a P1-regulated early promoter by the superimposition of a C1 operator. This combination of early- and late-promoter elements regulates and fine-tunes the expression of the particle maturation gene. During lysogenic growth the gene is turned off by P1 immunity functions. Upon induction of lytic growth, the expression of
mat
starts simultaneously with the expression of other C1-regulated P1 early functions. However, while most of the latter functions are downregulated during late stages of lytic growth the expression of
mat
continues throughout the entire lytic growth cycle of bacteriophage P1. Thus, the maturation function has a head start on the structural components of the phage particle.
Extragenic Suppressors of Growth Defects inmsbB Salmonella
Authors: Murray Sean R. , Bermudes David , de Felipe Karim Suwwan , Low K. Brooks
Published: July 2002

Lipid A, a potent endotoxin which can cause septic shock, anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of gram-negative bacteria. MsbB acylates (KDO)2-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis. Reports of knockouts of themsbBgene describe effects on virulence but describe no evidence of growth defects inEscherichia coliK-12 orSalmonella. Our data confirm the general lack of growth defects inmsbB E. coliK-12. In contrast,msbB Salmonella entericaserovar Typhimurium exhibits marked sensitivity to galactose-MacConkey and 6 mM EGTA media. At 37°C in Luria-Bertani (LB) broth,msbB Salmonellacells elongate, form bulges, and grow slowly.msbB Salmonellagrow well on LB-no salt (LB-0) agar; however, under specific shaking conditions in LB-0 broth, manymsbB Salmonellacells lyse during exponential growth and a fraction of the cells form filaments.msbB Salmonellagrow with a near-wild-type growth rate in MSB (LB-0 containing Mg2+and Ca2+) broth (23 to 42°C). Extragenic compensatory mutations, which partially suppress the growth defects, spontaneously occur at high frequency, and mutants can be isolated on media selective for faster growing derivatives. One of the suppressor mutations maps at 19.8 centisomes and is a recessive IS10insertional mutation insomA, a gene of unknown function which corresponds toybjXinE. coli. In addition, random Tn10mutagenesis carried out in an unsuppressedmsbBstrain produced a set of Tn10inserts, not inmsbBorsomA, that correlate with different suppressor phenotypes. Thus, insertional mutations, insomAand other genes, can suppress themsbBphenotype.
Chemical Structure of a Novel Aminophospholipid from
Hydrogenobacter thermophilus
Strain TK-6
Authors: Yoshino Jun-ichiro , Sugiyama Yasumasa , Sakuda Shohei , Kodama Tohru , Nagasawa Hiromichi , Ishii Masaharu , Igarashi Yasuo
Published: July 2002

The phospholipid composition of
Hydrogenobacter thermophilus
strain TK-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. Two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. An aminophospholipid named PX, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mass spectrometry (FAB-MS) and by analysis of the intact PX with FAB-MS as 1,2-diacyl-3-
O
-(phospho-2′-
O
-(1′-amino)-2′,3′,4′,5′-pentanetetrol)-sn-glycerol. Structurally similar phospholipids have been identified in
Methanospirillum hungatei
,
Methanolacinia paynteri
, and
Methanogenium cariaci
, which all belong to the
Archaea
.
Modulation of Gonococcal Piliation by Regulatable Transcription of
pilE
Authors: Long Cynthia D. , Hayes Stanley F. , van Putten Jos P. M. , Harvey Hillery A. , Apicella Michael A. , Seifert H. Steven
Published: July 2002

The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (Δ
pilE1
) in which the
lacIOP
regulatory sequences control
pilE
transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-β-
d
-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level of
pilE
transcription, without a change in
pilE
sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.
New Host-Vector System for
Thermus
spp. Based on the Malate Dehydrogenase Gene
Authors: Kayser Kevin J. , Kilbane John J.
Published: July 2002

A
Thermus thermophilus
HB27 strain was constructed in which the malate dehydrogenase
(mdh)
gene was deleted. The
Δmdh
colonies are recognized by a small-colony phenotype. Wild-type phenotype is restored by transformation with
Thermus
plasmids or integration vector containing an intact
mdh
gene. The wild-type phenotype provides a positive selection tool for the introduction of plasmid DNA into
Thermus
spp., and because
mdh
levels can be readily quantified, this host-vector system is a convenient tool for monitoring gene expression.