bluuebunny/crossref_metadata_2025_split huggingface dataset:
luxSandarcBControl Aerobic Growth ofActinobacillus actinomycetemcomitansunder Iron Limitation
Authors: Fong Karen P. , Gao Ling , Demuth Donald R. 
Published: December 2002

ABSTRACTLuxS is responsible for the production of autoinducer 2 (AI-2), which functions inVibrio harveyias a quorum-sensing signal that controls the cell density-dependent expression of theluxoperon. In nonluminescent organisms, the physiologic role of AI-2 is not clear. We report that inactivation ofluxSinActinobacillus actinomycetemcomitansJP2 results in reduced growth of the mutant, but not the wild-type organism, under aerobic, iron-limited conditions. Stunted cultures of theluxSmutantA. actinomycetemcomitansJP2-12 grew to high cell density when subcultured under iron-replete conditions. In addition, the mutant strain grew to high cell density under iron limitation after transformation with a plasmid containing a functional copy ofluxS.Results of real-time PCR showed thatA. actinomycetemcomitansJP2-12 exhibited significantly reduced expression ofafuA(eightfold),fecBCDE(10-fold), andftnAB(>50-fold), which encode a periplasmic ferric transport protein, a putative ferric citrate transporter, and ferritin, respectively. The expressions of putative receptors for transferrin, hemoglobin, and hemophore binding protein were also reduced at more modest levels (two- to threefold). In contrast, expressions ofsidDandfrpB(encoding putative siderophore receptors) were increased 10- and 3-fold, respectively, in theluxSmutant. To better understand the mechanism of the AI-2 response, theA. actinomycetemcomitansgenome was searched for homologs of theV. harveyisignal transduction proteins, LuxP, LuxQ, LuxU, and LuxO. Interestingly, ArcB was found to be most similar to LuxQ sensor/kinase. To determine whetherarcBplays a role in the response ofA. actinomycetemcomitansto AI-2, anarcB-deficient mutant was constructed. The isogenicarcBmutant grew poorly under anaerobic conditions but grew normally under aerobic iron-replete conditions. However, thearcBmutant failed to grow aerobically under iron limitation, and reverse transcriptase PCR showed that inactivation ofarcBresulted in decreased expression ofafuAandftnAB. Thus, isogenicluxSandarcBmutants ofA. actinomycetemcomitansexhibit similar phenotypes when cultured aerobically under iron limitation, and both mutants exhibit reduced expression of a common set of genes involved in the transport and storage of iron. These results suggest that LuxS and ArcB may act in concert to control the adaptation ofA. actinomycetemcomitansto iron-limiting conditions and its growth under such conditions.
Expression ofl-Selectin (CD62L), CD44, and CD25 on Activated Bovine T Cells
Authors: Waters W. R. , Rahner T. E. , Palmer M. V. , Cheng D. , Nonnecke B. J. , Whipple D. L. 
Published: December 2002

ABSTRACTMycobacterium bovisinfection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects ofM. bovisinfection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells fromM. bovis-infected cattle were stimulated withM. bovispurified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4+cells from infected animals. CD62L MFI on PPD- and PWM-stimulated γδ T-cell receptor-positive (TCR+) and CD8+cells was also reduced compared to that of nonstimulated γδ TCR+and CD8+cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4+cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs ofM. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.
Antiviral Activity of Shiga Toxin Requires Enzymatic Activity and Is Associated with Increased Permeability of the Target Cells
Authors: Basu Indira , Ferens Witold A. , Stone Diana M. , Hovde Carolyn J. 
Published: December 2002

ABSTRACTThis study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA11 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producingEscherichia colicolonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.
Effective Protective Immunity toYersinia pestisInfection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen
Authors: Grosfeld Haim , Cohen Sara , Bino Tamar , Flashner Yehuda , Ber Raphael , Mamroud Emanuelle , Kronman Chanoch , Shafferman Avigdor , Velan Baruch 
Published: December 2002

ABSTRACTThree plasmids expressing derivatives of theYersinia pestiscapsular F1 antigen were evaluated for their potential as DNA vaccines. These included plasmids expressing the full-length F1, F1 devoid of its putative signal peptide (deF1), and F1 fused to the signal-bearing E3 polypeptide of Semliki Forest virus (E3/F1). Expression of these derivatives in transfected HEK293 cells revealed that deF1 is expressed in the cytosol, E3/F1 is targeted to the secretory cisternae, and the nonmodified F1 is rapidly eliminated from the cell. Intramuscular vaccination of mice with these plasmids revealed that the vector expressing deF1 was the most effective in eliciting anti-F1 antibodies. This response was not limited to specific mouse strains or to the mode of DNA administration, though gene gun-mediated vaccination was by far more effective than intramuscular needle injection. Vaccination of mice with deF1 DNA conferred protection against subcutaneous infection with the virulentY. pestisKimberley53 strain, even at challenge amounts as high as 4,000 50% lethal doses. Antibodies appear to play a major role in mediating this protection, as demonstrated by passive transfer of anti-deF1 DNA antiserum. Taken together, these observations indicate that a tailored genetic vaccine based on a bacterial protein can be used to confer protection against plague in mice without resorting to regimens involving the use of purified proteins.
Interaction of Ler at the
LEE5
(
tir
) Operon of Enteropathogenic
Escherichia coli
Authors: Haack Kenneth R. , Robinson Christopher L. , Miller Kristie J. , Fowlkes Jonathan W. , Mellies Jay L. 
Published: December 2002

ABSTRACT

The genome of enteropathogenic
Escherichia coli
(EPEC) encodes a global regulator, Ler (locus of enterocyte effacement [LEE]-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the
LEE2
-
LEE3
divergent operon pair containing overlapping −10 regions and the
LEE5
(
tir
) operon. Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS. In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the
LEE5
operon. We determined the
cis
-acting DNA sequences necessary for Ler binding at
LEE5
by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at
LEE5
by binding sequences between positions −190 and −73 in relation to the transcriptional start site. Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at
LEE5
, we hypothesized that multiple Ler proteins bind upstream of the
LEE5
promoter to increase transcriptional activity from a distance. Using an
hns
deletion strain, we demonstrated that like the
LEE2
-
LEE3
operon pair, H-NS represses
LEE5
transcription. We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex.
CD36-Mediated Nonopsonic Phagocytosis of Erythrocytes Infected with Stage I and IIA Gametocytes of
Plasmodium falciparum
Authors: Smith Todd G. , Serghides Lena , Patel Samir N. , Febbraio Maria , Silverstein Roy L. , Kain Kevin C. 
Published: December 2002

ABSTRACT

Gametocytes, the sexual stages of malaria parasites (
Plasmodium
spp.) that are transmissible to mosquitoes, have been the focus of much recent research as potential targets for novel drug and vaccine therapies. However, little is known about the host clearance of gametocyte-infected erythrocytes (GEs). Using a number of experimental strategies, we found that the scavenger receptor CD36 mediates the uptake of nonopsonized erythrocytes infected with stage I and IIA gametocytes of
Plasmodium falciparum
by monocytes and culture-derived macrophages (Mφs). Light microscopy and immunofluorescence assays revealed that stage I and IIA gametocytes were readily internalized by monocytes and Mφs. Pretreating monocytes and Mφs with a monoclonal antibody that blocked CD36 resulted in a significant reduction in phagocytosis, as did treating GEs with low concentrations of trypsin to remove
P. falciparum
erythrocyte membrane protein 1 (PfEMP-1), a parasite ligand for CD36. Pretreating monocytes and Mφs with peroxisome proliferator-activated receptor γ-retinoid X receptor agonists, which specifically upregulate CD36, resulted in a significant increase in the phagocytosis of GEs. Murine CD36 on mouse Mφs also mediated the phagocytosis of
P. falciparum
stage I and IIA gametocytes, as determined by receptor blockade with anti-murine CD36 monoclonal antibodies and the lack of uptake by CD36-null Mφs. These results indicate that phagocytosis of stage I and IIA gametocytes by monocytes and Mφs appears to be mediated to a large extent by the interaction of PfEMP-1 and CD36, suggesting that CD36 may play a role in innate clearance of these early sexual stages.
Mucosal Immunization withHelicobacter, CpG DNA, and Cholera Toxin Is Protective
Authors: Jiang Weiwen , Baker Henry J. , Smith Bruce F. 
Published: December 2002

ABSTRACTThe mucosal delivery of antigens requires an effective adjuvant to induce mucosal immunity. Current mucosal adjuvants include cholera toxin (CT) andEscherichia coliheat-labile toxin. Unmethylated CpG immunostimulatory oligodeoxynucleotides (ODNs) have been proposed as novel mucosal adjuvants. In this study, mice were immunized with sonicatedHelicobacter feliswith CT and/or CpG ODN adjuvants. All groups receiving either adjuvant singly or in combination developed increased serum anti-H. felisimmunoglobulin G (IgG). The addition of either CpG or CT, or both, produced a specific fecal anti-H. felisIgA response, with the highest IgA levels occurring in animals immunized intranasally with sonicatedH. feliswith CT and CpG. FollowingH. felischallenge, addition of the adjuvant CpG ODN provided no significant protection, while groups given CT showed a high degree of protection, although not complete. When CpG ODN was combined with CT and the vaccine combination was delivered intranasally, no bacterial colonization was detected by quantitative PCR, providing “sterile immunity” and demonstrating synergy between CpG ODN and CT.
Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis
Authors: Ahmed Saeed , Colmenares M. , Soong L. , Goldsmith-Pestana K. , Munstermann L. , Molina R. , McMahon-Pratt Diane 
Published: December 2002

ABSTRACTThe levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4+and CD8+T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing theLeishmania infantumD-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.
Roles of the Maltese Cross Form in the Development of Parasitemia and Protection against
Babesia microti
Infection in Mice
Authors: Yokoyama Naoaki , Bork Sabine , Nishisaka Mitsuhiro , Hirata Haruyuki , Matsuo Tomohide , Inoue Noboru , Xuan Xuenan , Suzuki Hiroshi , Sugimoto Chihiro , Igarashi Ikuo 
Published: December 2002

ABSTRACT

Babesia microti,
a hemoprotozoan parasite of rodents, is also important as a zoonotic agent of human babesiosis. The Maltese cross form, which consists of four masses in an erythrocyte, is characteristic of the developmental stage of
B. microti
. Monoclonal antibody (MAb) 2-1E, which specifically recognizes the Maltese cross form of
B. microti
, has been described previously. In the present study, we examined the roles of the Maltese cross form during the infectious course of
B. microti
in mice. The number of the Maltese cross form increased in the peripheral blood of infected mice prior to the peak of parasitemia. With confocal laser scanning microscopy, MAb 2-1E was found to be reactive with the ring form, with the parasites undergoing transformation to the Maltese cross form and subsequent division, and also with extracellular merozoites. Furthermore, the Maltese cross form-related antigen (MRA) gene was isolated from a
B. microti
cDNA library by immunoscreening with MAb 2-1E, and the nucleotide sequence was determined. Genomic analyses indicated that the MRA gene exists as a single-copy gene in
B. microti
. Immunization of mice with recombinant MRA induced significant protective immunity against
B. microti
infection. These findings indicate that the Maltese cross form plays important roles in both the development of parasitemia and the protective response against the infection.
Normal Host Defense during Systemic Candidiasis in Mannose Receptor-Deficient Mice
Authors: Lee Sena J. , Zheng Nai-Ying , Clavijo Monica , Nussenzweig Michel C. 
Published: December 2002

ABSTRACTPathogen pattern recognition receptors (PRRs) recognize common structural and molecular motifs present on microbial surfaces and contribute to induction of innate immune responses. The mannose receptor (MR), a carbohydrate-binding receptor expressed on subsets of macrophages, is considered one such PRR. In vitro experiments have implicated the MR in phagocytosis of mannose-bearing microbes, includingCandida albicans, and enhancement of antifungal response by macrophages. However, the significance of the MR's contribution to immune response during systemicC. albicansinfection has never been directly demonstrated. Using MR-deficient mice in an in vivo infection experiment, we examined the role of the MR in immune response during disseminated candidiasis. MR−/−and wild-type control mice were challenged intraperitoneally withC. albicans, and the survival rates, tissue fungal burden, inflammatory cell recruitment, and specific antibody production after infection were evaluated. We found no significant difference in survival between the two mouse strains. MR−/−mice had higher average fungal burdens in some of the organs on days 7 and 21 but exhibited competence in inflammatory cell recruitment and antibody production. We also observed in vitro that MR−/−peritoneal cavity macrophages were equally capable ofC. albicansuptake and that phagocytosis could be blocked with β-glucan. We conclude that the MR is not required for the normal host defense during disseminated candidiasis or for the phagocytosis ofC. albicansand that a β-glucan receptor may be required forC. albicansphagocytosis.
Cytotoxic Effects of Streptolysin O and Streptolysin S Enhance the Virulence of Poorly Encapsulated Group A Streptococci
Authors: Sierig Gabriele , Cywes Colette , Wessels Michael R. , Ashbaugh Cameron D. 
Published: December 2002

ABSTRACTAlthough the toxicity of streptolysin O (SLO) and streptolysin S (SLS) in purified group A streptococci (GAS) has been established, the effect of these molecules in natural infection is not well understood. To identify whether biologically relevant concentrations of SLO and SLS were cytotoxic to epithelial and phagocytic cells that the bacteria would typically encounter during human infection and to characterize the influence of cell injury on bacterial pathogenesis, we derived GAS strains deficient in SLO or SLS in the background of an invasive GAS M3 isolate and determined their virulence in in vitro and in vivo models of human disease. Whereas bacterial production of SLO resulted in lysis of both human keratinocytes and polymorphonuclear leukocytes, GAS expression of SLS was associated only with keratinocyte injury. Expression of SLO but not SLS impaired polymorphonuclear leukocyte killing of GAS in vitro, but this effect could only be demonstrated in the background of acapsular organisms. In mouse invasive soft-tissue infection, neither SLO or SLS expression significantly influenced mouse survival. By contrast, in a mouse model of bacterial sepsis after intraperitoneal inoculation of GAS, SLO expression enhanced the virulence of both encapsulated and acapsular GAS, whereas SLS expression increased the virulence only of acapsular GAS. We conclude that the cytotoxic effects of SLO protect GAS from phagocytic killing and enhance bacterial virulence, particularly of strains that may be relatively deficient in hyaluronic acid capsule. Compared to SLO, SLS in this strain background has a more modest influence on GAS pathogenicity and the effect does not appear to involve bacterial resistance to phagocytosis.
Inhibition of
Mycobacterium tuberculosis
Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo
Authors: Harth Günter , Horwitz Marcus A. 
Published: December 2002

ABSTRACT

Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent,
Mycobacterium tuberculosis
, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting
M. tuberculosis
glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibiotic strategy. In guinea pigs challenged by aerosol with the highly virulent Erdman strain of
M. tuberculosis
, the GS inhibitor
l
-methionine-
SR
-sulfoximine (MSO) protected the animals against weight loss, a hallmark of tuberculosis, and against the growth of
M. tuberculosis
in the lungs and spleen; MSO reduced the CFU of
M. tuberculosis
at 10 weeks after challenge by ∼0.7 log unit compared with that in control animals. MSO acted synergistically with isoniazid in protecting animals against weight loss and bacterial growth, reducing the CFU in the lungs and spleen by ∼1.5 log units below the level seen with isoniazid alone. In the presence of ascorbate, which allows treatment with a higher dose, MSO was highly efficacious, reducing the CFU in the lungs and spleen by 2.5 log units compared with that in control animals. This study demonstrates that inhibition of
M. tuberculosis
GS is a feasible therapeutic strategy against this pathogen and supports the concept that
M. tuberculosis
enzymes involved in cell wall biosynthesis, including major secretory proteins, have potential as antibiotic targets.
Identification of LpeA, a PsaA-Like Membrane Protein That Promotes Cell Entry byListeria monocytogenes
Authors: Réglier-Poupet Hélène , Pellegrini Elisabeth , Charbit Alain , Berche Patrick 
Published: December 2002

ABSTRACTThe intracellular life ofListeria monocytogenesstarts by a complex process of entry involving several bacterial ligands and eukaryotic receptors. In this work, we identified in silico from the sequence of the genome ofL. monocytogenesa previously unknown gene designatedlpeA(for lipoprotein promoting entry) encoding a 35-kDa protein homologous to PsaA, a lipoprotein belonging to the LraI family and implicated in the cell adherence ofStreptococcus pneumoniaeand related species. By constructing a mutant ofL. monocytogenesin whichlpeAis deleted (lpeAmutant), we show that the PsaA-like protein LpeA is not involved in bacterial adherence but is required for entry ofL. monocytogenesin eukaryotic cells. In contrast to wild-type bacteria, mutant bacteria failed to invade the epithelial Caco-2 and hepatocyte TIB73 cell lines, as confirmed by confocal microscopy. The mutant bacteria rapidly penetrated in mouse bone marrow-derived macrophages. Surprisingly,lpeAmutant bacteria survive better in macrophages than do wild-type bacteria. This was correlated with a weak exacerbation of virulence of thelpeAmutant in the mouse. LpeA is therefore a novel invasin favoring the entry ofL. monocytogenesinto nonprofessional phagocytes but not its invasion of macrophages. This is the first report of a lipoprotein promoting cell invasion of an intracellular pathogen.