bluuebunny/crossref_metadata_2025 huggingface dataset:
Cleavage of Antigen-Bound Immunoglobulin G by SpeB Contributes to Streptococcal Persistence in Opsonizing Blood
Authors: Eriksson Anna , Norgren Mari
Published: December 2002

Group A streptococci (GAS) express a superantigen, SpeB, having cysteine protease activity. SpeB exhibits several properties that might contribute to virulence, the most recently discovered being the ability to cleave immunoglobulin G (IgG) in a manner similar to that of papain. In the present study, we confirmed this latter finding and found that the irreversible inhibition of SpeB protease activity completely abolishes IgG cleavage. SpeB cleavage of IgG was not species restricted since SpeB cleaved both human, rabbit, and mouse IgG. In order to investigate the nature of the SpeB cleavage of IgG, antibodies were immobilized prior to exposure to SpeB, either by unspecific binding of the Fc to GAS surface proteins or by antigen-specific binding. Analysis of the IgG molecules by SDS-PAGE showed that SpeB could cleave antigen-bound antibodies, while the IgG bound to IgG-binding proteins was protected from cleavage. In a phagocytosis assay using whole blood, the M49 GAS strain NZ131 showed a significantly higher survival than its isogenicspeBmutant. Furthermore, the addition of extracellular supernatant derived from an overnight culture of native NZ131 increased the survival of its isogenicspeBderivative. This indicates that SpeB's ability to cleave off the Fc part of antigen-bound IgG contributes to GAS escape from opsonophagocytosis while not interfering with the formation of a host-like coat by unspecific IgG binding.
Genetic Alteration of Capsule Type but Not PspA Type Affects Accessibility of Surface-Bound Complement and Surface Antigens ofStreptococcus pneumoniae
Authors: Abeyta Melanie , Hardy Gail G. , Yother Janet
Published: December 2002

TheStreptococcus pneumoniaecapsular polysaccharides and pneumococcal surface protein A (PspA) are major determinants of virulence that are antigenically variable and capable of eliciting protective immune responses. By genetically switching the pspA genes of the capsule type 2 strain D39 and the capsule type 3 strain WU2, we showed that the different abilities of antibody to PspA to protect against these strains was not related to the PspA type expressed. Similarly, the level of specific antibody binding to PspA, other surface antigens, and surface-localized C3b did not depend on the PspA type but instead was correlated with the capsule type. The type 3 strain WU2 and an isogenic derivative of D39 that expresses the type 3 capsule bound nearly identical amounts of antibody to PspA and other surface antigens, and these amounts were less than one-half the amount observed with the type 2 parent strain D39. Expression of the type 3 capsule in D39 also reduced the amount of C3b deposited and its accessibility to antibody, resulting in a level intermediate between the levels observed with WU2 and D39. Despite these effects, the capsule type was not the determining factor in anti-PspA-mediated protection, as both D39 and its derivative expressing the type 3 capsule were more resistant to protection than WU2. The specific combination of PspA and capsule type also did not determine the level of protection. The capsule structure is thus a major determinant in accessibility of surface antigens to antibody, but certain strains appear to express other factors that can influence antibody-mediated protection.
Resistance of Macrophages toMycobacterium aviumIs Induced by α2-Adrenergic Stimulation
Authors: Weatherby Kelly E. , Zwilling Bruce S. , Lafuse William P.
Published: December 2002

The ability of macrophages to control the growth of microorganisms is increased by macrophage activation. Previously, it was shown that epinephrine activated mouse macrophages to resist the growth ofMycobacterium aviumvia α2-adrenergic stimulation. In the present study, we show that the α2-adrenergic agonist (α2-agonist) clonidine induced resistance toM. aviumgrowth in the RAW264.7 mouse macrophage cell line. The ability of catecholamines to induce resistance to mycobacteria was specific to α2-adrenergic stimulation, as α1-, β1-, and β2-agonists had no effect. Receptor signaling through Gi proteins was required. A G-protein antagonist specific for the α subunits of the Go/Gi family blocked the increased resistance induced by clonidine, while a Gs-protein antagonist was without effect. Both nitric oxide (NO) production and superoxide (O2−) production were required for the increased resistance toM. aviumgrowth induced by clonidine. Although NO production was required, clonidine did not increase the level of NO inM. avium-infected cells. Since NO and O2−interact to produce peroxynitrite (ONOO−), we examined whether ONOO−mediates the increased resistance toM. aviuminduced by clonidine. 5,10,15,20-Tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride (FeTPPS), a specific scavenger of ONOO−, inhibited the effect of clonidine onM. aviumgrowth. Clonidine also increased the production of ONOO−inM. avium-infected RAW264.7 cells, as measured by the oxidation of 123-dihydrorhodamine and the production of nitrated tyrosine residues. We therefore conclude that α2-adrenergic stimulation activates macrophages to resist the growth ofM. aviumby enhancing the production of ONOO−.
Induction of Osteoclastogenesis and Matrix Metalloproteinase Expression by the Lipooligosaccharide ofTreponema denticola
Authors: Choi Bong-Kyu , Lee Hyun Jung , Kang Jung Hwa , Jeong Gook Jin , Min Cheon Ki , Yoo Yun-Jung
Published: December 2002

Alveolar bone destruction is a characteristic feature of periodontitis.Treponema denticolais known to be involved in periodontitis. To elucidate the role ofT. denticolain alveolar bone destruction in periodontitis, the effects of lipooligosaccharide (LOS) fromT. denticolaon osteoclast formation and on expression of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) mRNAs were examined in a coculture system by using mouse calvaria and bone marrow cells. In addition, the effect ofT. denticolaLOS on expression of matrix metalloproteinases (MMPs), which are involved in bone resorption, was estimated in mouse calvaria-derived osteoblastic cells. When the mouse calvaria and bone marrow cells were challenged with LOS (0.1 to 10 μg/ml) for 4 days, the number of tartrate-resistant acid phosphatase-positive multinucleated cells increased in a dose-dependent manner. The expression of ODF mRNA increased, while OPG mRNA expression decreased. Polymyxin B changed the effect of LOS (10 μg/ml) on ODF and OPG mRNA expression to the control level. LOS (10 μg/ml) stimulated prostaglandin E2(PGE2) production in the cocultures. Adding indomethacin, an inhibitor of prostaglandin synthesis, resulted in a reduction in the number of osteoclasts induced by LOS and eliminated the effect ofT. denticolaLOS on ODF and OPG mRNA expression.T. denticolaLOS increased the levels of mRNAs encoding MMP-3, -8, -9, -10, -13, and -14. Expression of one of these mRNAs, MMP-9 mRNA, was significantly induced byT. denticolaLOS. These findings suggest that LOS fromT. denticolastimulates osteoclastogenesis and MMP expression. Up-regulation of ODF and down-regulation of OPG by a PGE2-dependent mechanism were involved in the osteoclastogenesis induced byT. denticolaLOS.
Seeligeriolysin O, a Cholesterol-Dependent Cytolysin ofListeria seeligeri, Induces Gamma Interferon from Spleen Cells of Mice
Authors: Ito Yutaka , Kawamura Ikuo , Kohda Chikara , Baba Hisashi , Nomura Takamasa , Kimoto Terumi , Watanabe Isao , Mitsuyama Masao
Published: December 2002

Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced byListeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced byListeria monocytogenesat the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-γ) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-γ production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-γ production but also LSO-induced IFN-γ production. Both NK cells and CD11b+cells were required for LSO-induced IFN-γ production. Among the various cytokines expressed in CD11b+cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.
Placental Tumor Necrosis Factor Alpha but Not Gamma Interferon Is Associated with Placental Malaria and Low Birth Weight in Malawian Women
Authors: Rogerson Stephen J. , Brown Heidi C. , Pollina Elena , Abrams Elizabeth T. , Tadesse Eyob , Lema Valentino M. , Molyneux Malcolm E.
Published: December 2002

Malaria in pregnancy predisposes to maternal anemia and low birth weight (LBW). We examined the possible roles of the cytokines tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) in these adverse outcomes. We measured cytokine concentrations in placental, peripheral, and cord blood plasma in relation to malaria parasitemia and placental monocyte accumulation in 276 Malawian women. Maternal hemoglobin concentration, human immunodeficiency virus status, and infant birth weight were determined. Concentrations of TNF-α in placental blood were correlated with densities ofPlasmodium falciparum-infected erythrocytes (P< 0.0001) and of intervillous monocyte infiltrates (P< 0.0001) on placental histology. Peripheral blood TNF-α concentrations were relatively low and were weakly associated with malaria. TNF-α concentrations were higher in placental blood, where they were strongly associated with malaria. Placental plasma TNF-α levels were higher in women who had LBW babies (P= 0.0027), women with febrile symptoms (P< 0.0001), and teenage mothers (P= 0.04) than in other women. The presence of TNF-α in cord blood was not associated with malaria infection. IFN-γ levels were infrequently elevated, and elevated IFN-γ levels were not associated with poor pregnancy outcomes. Placental production of TNF-α, but not of IFN-γ, may be implicated in impaired fetal growth in Malawian women.
Age-Related Disparity in Functional Activities of Human Group C Serum Anticapsular Antibodies Elicited by Meningococcal Polysaccharide Vaccine
Authors: Harris Shannon L. , King W. James , Ferris Wendy , Granoff Dan M.
Published: December 2002

Serum bactericidal activity confers protection against meningococcal disease, but it is not known whether vaccine-induced anticapsular antibodies that lack bactericidal activity are protective. We developed an infant rat challenge model using a naturally occurring O-acetylated strain ofNeisseria meningitidisgroup C and a strain that was negative for O acetylation (OAc). Rats 4 to 7 days of age inoculated intraperitoneally (i.p.) with ∼103CFU of either strain developed >5 × 105CFU/ml of blood obtained 18 h later. Dilutions of preimmunization sera given i.p. 2 h before the bacterial challenge had no effect on bacteremia, whereas group C anticapsular antibody in sera from adults immunized with meningococcal polysaccharide vaccine conferred complete or partial (>99% decrease in CFU per milliliter of blood) protection against the OAc-positive or OAc-negative strain, respectively, at antibody doses as low as 0.04 μg/rat. Anticapsular antibody at doses fivefold higher (0.18 to 0.2 μg/rat) in pooled sera from children immunized at a mean age of 2.6 years failed to protect rats, but antibody at the same or fivefold-lower dose in a serum pool from a group of children immunized at 4 years of age gave complete or partial protection. Protective activity was observed with some serum pools that lacked detectable complement-mediated bactericidal activity (titers < 1:4) and correlated with increasing antibody avidity. Thus, not only does the magnitude of the group C antibody response to meningococcal polysaccharide vaccine increase with increasing age but there are also age-related affects on antibody functional activity such that higher serum concentrations of vaccine-induced antibody are required for protection of immunized children than for immunized adults.
Impact of Vector Priming on the Immunogenicity of RecombinantSalmonellaVaccines
Authors: Vindurampulle Christofer J. , Attridge Stephen R.
Published: December 2002

There are conflicting reports concerning the impact of prior vector priming on the immunogenicity of recombinant-Salmonella-based vaccines. A comparison of experimental protocols identified two variables which might account for this inconsistency: the potential of the vector strain to colonize the murine gut-associated lymphoid tissue (GALT) and the nature of the foreign antigen subsequently delivered by the recombinantSalmonellaconstruct. The former was investigated by constructing anaroAmutant of theSalmonella entericaserovar Stanley vector previously used in our laboratory. Although the introduction of anaroAmutation had surprisingly little effect on GALT colonization, it did reduce the strength of antilipopolysaccharide (anti-LPS) antibody responses and the impact of vector priming. Studies were also performed to ascertain the extent to which any observed hyporesponsiveness consequent upon vector priming might be determined by the characteristics of the foreign antigen.S. entericaserovar Stanley was used to deliver either of twoEscherichia coliantigens, K88 pilus protein or the LT-B toxin subunit, to vector-primed mice. Both serum immunoglobulin G (IgG) and intestinal IgA responses to K88 were completely abolished, and those to LT-B were significantly reduced, as a consequence of vector priming. When similar experiments were performed with anaroA S. entericaserovar Dublin vector, responses to K88 were significantly reduced but those to LT-B were unaffected by vector priming. Paradoxically, a priming infection with this vector induced stronger anti-LPS antibody responses but was less likely to elicit a state of hyporesponsiveness to subsequently presented foreign antigen. The impact of vector priming thus depends on both theSalmonellastrain used and the nature of the foreign antigen, but our present data strengthen concerns that preexisting antivector immunity represents a serious threat to theSalmonella-based vaccine strategy.
luxSandarcBControl Aerobic Growth ofActinobacillus actinomycetemcomitansunder Iron Limitation
Authors: Fong Karen P. , Gao Ling , Demuth Donald R.
Published: December 2002

LuxS 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

Mycobacterium 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

This 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

Three 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

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

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

The 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

The 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

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

Pathogen 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

Although 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

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

The 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.
Mapping of Murine Th1 Helper T-Cell Epitopes of Mycolyl Transferases Ag85A, Ag85B, and Ag85C fromMycobacterium tuberculosis
Authors: D'Souza S. , Rosseels V. , Romano M. , Tanghe A. , Denis O. , Jurion F. , Castiglione N. , Vanonckelen A. , Palfliet K. , Huygen Kris
Published: December 2002

BALB/c (H-2d) and C57BL/6 (H-2b) mice were infected intravenously withMycobacterium tuberculosisH37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C fromM. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-γ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A inM. bovisBCG-vaccinated and tuberculosis-infectedH-2dhaplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.
Cholera Toxin Induces Migration of Dendritic Cells from the Subepithelial Dome Region to T- and B-Cell Areas of Peyer's Patches
Authors: Shreedhar Vijay K. , Kelsall Brian L. , Neutra Marian R.
Published: December 2002

Intestinal M cells deliver macromolecules, particles, and pathogens into the subepithelial dome (SED) region of Peyer's patch mucosa, an area rich in dendritic cells (DCs). We tested whether uptake of the mucosal adjuvant cholera toxin (CT) or liveSalmonellabacteria can induce DC migration within Peyer's patches. Virus-sized, fluorescent polystyrene microparticles were efficiently transported by M cells and ingested by CD11c+, CD11b−, and CD8a−DCs in the SED region. DCs loaded with microparticles remained in the SED for up to 14 days. CT (but not the CT B subunit) and live attenuatedSalmonella entericaserovar Typhimurium bacteria induced migration of the microparticle-loaded DCs from the SED region into underlying B-cell follicles and adjacent parafollicular T-cell zones. Our data provide the first demonstration that DCs move in response to enterotoxin adjuvants and live bacteria that enter the mucosa via M cells.
Role of
sarA
in the Pathogenesis of
Staphylococcus aureus
Musculoskeletal Infection
Authors: Blevins Jon S. , Elasri Mohamed O. , Allmendinger Scott D. , Beenken Karen E. , Skinner Robert A. , Thomas J. Roby , Smeltzer Mark S.
Published: December 2002

We recently demonstrated that mutation of
sarA
in clinical isolates of
Staphylococcus aureus
results in a phenotype that is distinct by comparison to
sarA
mutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70:470-480, 2002). This raises the possibility that studies demonstrating that RN6390
sarA
mutants are attenuated do not accurately reflect the role of
sarA
in the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of
sarA
and
agr
mutants generated in a clinical isolate of
S. aureus
(UAMS-1). By using this model, we confirmed that mutation of
sarA
and/or
agr
results in a reduced capacity to cause both septic arthritis and osteomyelitis.
Production of Macrophage Inflammatory Protein 3α (MIP-3α) (CCL20) and MIP-3β (CCL19) by Human Peripheral Blood Neutrophils in Response to Microbial Pathogens
Authors: Akahoshi Tohru , Sasahara Takeshi , Namai Rie , Matsui Toshimichi , Watabe Hiroyuki , Kitasato Hidero , Inoue Matsuhisa , Kondo Hirobumi
Published: December 2002

Effects of bacterial pathogens on the production of macrophage inflammatory protein 3α (MIP-3α) and MIP-3β from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.
Role of Virulence Factors in Resistance of Avian Pathogenic
Escherichia coli
to Serum and in Pathogenicity
Authors: Mellata Melha , Dho-Moulin Maryvonne , Dozois Charles M. , Curtiss Roy , Brown Peter K. , Arné Pascal , Brée Annie , Desautels Clarisse , Fairbrother John M.
Published: December 2002

In chickens, colibacillosis is caused by avian pathogenic
Escherichia coli
(APEC) via respiratory tract infection. Many virulence factors, including type 1 (F1A) and P (F11) fimbriae, curli, aerobactin, K1 capsule, and temperature-sensitive hemagglutinin (Tsh) and plasmid DNA regions have been associated with APEC. A strong correlation between serum resistance and virulence has been demonstrated, but roles of virulence factors in serum resistance have not been well elucidated. By using mutants of APEC strains TK3, MT78, and χ7122, which belong to serogroups O1, O2, and O78, respectively, we investigated the role of virulence factors in resistance to serum and pathogenicity in chickens. Our results showed that serum resistance is one of the pathogenicity mechanisms of APEC strains. Virulence factors that increased bacterial resistance to serum and colonization of internal organs of infected chickens were O78 lipopolysaccharide of
E. coli
χ7122 and the K1 capsule of
E. coli
MT78. In contrast, curli, type 1, and P fimbriae did not appear to contribute to serum resistance. We also showed that the
iss
gene, which was previously demonstrated to increase resistance to serum in certain
E. coli
strains, is located on plasmid pAPEC-1 of
E. coli
χ7122 but does not play a major role in resistance to serum for strain χ7122.
MprF-Mediated Lysinylation of Phospholipids in
Staphylococcus aureus
Leads to Protection against Oxygen-Independent Neutrophil Killing
Authors: Kristian Sascha A. , Dürr Manuela , Van Strijp Jos A. G. , Neumeister Birgid , Peschel Andreas
Published: December 2002

Staphylococcus aureus
achieves resistance to defensins and similar cationic antimicrobial peptides (CAMPs) by modifying anionic membrane lipids via MprF with
l
-lysine, which leads to repulsion of these host defense molecules.
S. aureus
Δ
mprF
, which lacks the modification, was very efficiently killed by neutrophil defensins and CAMP-producing leukocytes, even when oxygen-dependent killing was disrupted, but was as susceptible as wild-type bacteria to inactivation by myeloperoxidase or human monocytes lacking defensins. These results demonstrate the impact and specificity of MprF-mediated CAMP resistance and underscore the role of defensin-like peptides in innate host defense.
Involvement of
Trypanosoma cruzi
Metacyclic Trypomastigote Surface Molecule gp82 in Adhesion to Gastric Mucin and Invasion of Epithelial Cells
Authors: Neira Ivan , Silva Fernando A. , Cortez Mauro , Yoshida Nobuko
Published: December 2002

Upon oral infection,
Trypanosoma cruzi
metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other
T. cruzi
surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by metacyclic forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of metacyclic forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.
Enhanced Immunogenicity to
Mycobacterium tuberculosis
by Vaccination with an Alphavirus Plasmid Replicon Expressing Antigen 85A
Authors: Kirman Joanna R. , Turon Tara , Su Hua , Li Amy , Kraus Carl , Polo John M. , Belisle John , Morris Sheldon , Seder Robert A.
Published: December 2002

The immunogenicity of a plasmid DNA vaccine incorporating Sindbis virus RNA replicase functions (pSINCP) and expressing antigen 85A (Ag85A) from
Mycobacterium tuberculosis
was compared with a conventional plasmid DNA vector encoding Ag85A. pSINCP-85A was highly immunogenic in mice and gave enhanced long-term protection against
M. tuberculosis
compared with the conventional vector.