bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Human Antibodies to Erythrocyte Binding Antigen 175 of
Plasmodium falciparum
Authors: Okenu Daniel M. N. , Riley Eleanor M. , Bickle Quentin D. , Agomo Philip U. , Barbosa Arnoldo , Daugherty Jon R. , Lanar David E. , Conway David J.
Published: July 2002

Invasion of human erythrocytes by
Plasmodium falciparum
merozoites is a multistep process. For many strains of the parasite, part of this process requires that the erythrocyte binding antigen 175 (EBA-175) of the merozoite binds to sialic acid residues of glycophorin A on the erythrocyte surface, a receptor-ligand interaction which represents a potential target for inhibition by antibodies. This study characterizes the reactivity of naturally acquired human antibodies with four recombinant proteins representing parts of EBA-175 (region II, regions III to V, and the dimorphic C and F segment region) in populations in which the organism is endemic. Serum immunoglobulin G (IgG) recognizing the recombinant proteins is predominantly of the IgG1 and IgG3 subclasses, and its prevalence increases with age. In a large population study in The Gambia, serum positivity for IgG or IgG1 and IgG3 subclass antibodies to each of the EBA-175 recombinant antigens was not significantly associated with subsequent protection from clinical malaria. However, there was a trend indicating that individuals with high levels of IgG to region II may have some protection.
Interleukin 12 as an Adjuvant to WI-1 Adhesin Immunization Augments Delayed-Type Hypersensitivity, Shifts the Subclass Distribution of Immunoglobulin G Antibodies, and Enhances Protective Immunity toBlastomyces dermatitidisInfection
Authors: Wüthrich Marcel , Finkel-Jiminez Beatriz E. , Klein Bruce S.
Published: July 2002

Cell-mediated immunity is pivotal in host resistance toBlastomyces dermatitidisinfection. Immunization of mice with the WI-1 adhesin enhances resistance against experimental pulmonary infection but elicits features of a mixed T-helper-cell immune response. Immune mice acquire delayed-type hypersensitivity (DTH) but also high titers of WI-1-specific immunoglobulin G1 (IgG1) and IgG2b, a result indicative of T-helper-2 cellular immunity. We report that interleukin-12, used as an adjuvant for WI-1 immunization, augments DTH, shifts the balance of the T-helper phenotype toward Th1, and enhances resistance toB. dermatitidisinfection.
Bacterial Induction of Beta Interferon in Mice Is a Function of the Lipopolysaccharide Component
Authors: Sing Andreas , Merlin Thomas , Knopf Hans-Peter , Nielsen Peter J. , Loppnow Harald , Galanos Chris , Freudenberg Marina A.
Published: July 2002

We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lpsd]) C57BL/10ScCr mice to produce beta interferon (IFN-β) when stimulated with bacteria. For this purpose, the IFN-β and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lpsn]; C57BL/10ScSn and BALB/c) andLpsd(C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-β in theLpsnmice but not in theLpsdmice. All gram-positive bacteria tested failed to induce significant amounts of IFN-β in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1α [IL-1α], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-β and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-β in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-β is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.
Comparative Genome Analysis of the Pathogenic Spirochetes
Borrelia burgdorferi
and
Treponema pallidum
Authors: Subramanian G. , Koonin Eugene V. , Aravind L.
Published: July 2002

A comparative analysis of the predicted protein sequences encoded in the complete genomes of
Borrelia burgdorferi
and
Treponema pallidum
provides a number of insights into evolutionary trends and adaptive strategies of the two spirochetes. A measure of orthologous relationships between gene sets, termed the orthology coefficient (OC), was developed. The overall OC value for the gene sets of the two spirochetes is about 0.43, which means that less than one-half of the genes show readily detectable orthologous relationships. This emphasizes significant divergence between the two spirochetes, apparently driven by different biological niches. Different functional categories of proteins as well as different protein families show a broad distribution of OC values, from near 1 (a perfect, one-to-one correspondence) to near 0. The proteins involved in core biological functions, such as genome replication and expression, typically show high OC values. In contrast, marked variability is seen among proteins that are involved in specific processes, such as nutrient transport, metabolism, gene-specific transcription regulation, signal transduction, and host response. Differences in the gene complements encoded in the two spirochete genomes suggest active adaptive evolution for their distinct niches. Comparative analysis of the spirochete genomes produced evidence of gene exchanges with other bacteria, archaea, and eukaryotic hosts that seem to have occurred at different points in the evolution of the spirochetes. Examples are presented of the use of sequence profile analysis to predict proteins that are likely to play a role in pathogenesis, including secreted proteins that contain specific protein-protein interaction domains, such as von Willebrand A, YWTD, TPR, and PR1, some of which hitherto have been reported only in eukaryotes. We tentatively reconstruct the likely evolutionary process that has led to the divergence of the two spirochete lineages; this reconstruction seems to point to an ancestral state resembling the symbiotic spirochetes found in insect guts.
Identification of Potential Vaccine and Drug Target Candidates by Expressed Sequence Tag Analysis and Immunoscreening ofOnchocerca volvulusLarval cDNA Libraries
Authors: Lizotte-Waniewski Michelle , Tawe Wilson , Guiliano David B. , Lu Wenhong , Liu Jing , Williams Steven A. , Lustigman Sara
Published: July 2002

The search for appropriate vaccine candidates and drug targets against onchocerciasis has so far been confronted with several limitations due to the unavailability of biological material, appropriate molecular resources, and knowledge of the parasite biology. To identify targets for vaccine or chemotherapy development we have undertaken two approaches. First, cDNA expression libraries were constructed from life cycle stages that are critical for establishment ofOnchocerca volvulusinfection, the third-stage larvae (L3) and the molting L3. A gene discovery effort was then initiated by random expressed sequence tag analysis of 5,506 cDNA clones. Cluster analyses showed that many of the transcripts were up-regulated and/or stage specific in either one or both of the cDNA libraries when compared to the microfilariae, L2, and both adult stages of the parasite. Homology searches against the GenBank database facilitated the identification of several genes of interest, such as proteinases, proteinase inhibitors, antioxidant or detoxification enzymes, and neurotransmitter receptors, as well as structural and housekeeping genes. OtherO. volvulusgenes showed homology only to predicted genes from the free-living nematodeCaenorhabditis elegansor were entirely novel. Some of the novel proteins contain potential secretory leaders. Secondly, by immunoscreening the molting L3 cDNA library with a pool of human sera from putatively immune individuals, we identified six novel immunogenic proteins that otherwise would not have been identified as potential vaccinogens using the gene discovery effort. This study lays a solid foundation for a better understanding of the biology ofO. volvulusas well as for the identification of novel targets for filaricidal agents and/or vaccines against onchocerciasis based on immunological and rational hypothesis-driven research.
Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family ofEscherichia coliThat Infect the Human Polarized Intestinal Caco-2/TC7 Cells
Authors: Guignot Julie , Peiffer Isabelle , Bernet-Camard Marie-Françoise , Lublin Douglas M. , Carnoy Christophe , Moseley Steve L. , Servin Alain L.
Published: July 2002

The Afa/Dr family of diffusely adheringEscherichia coli(Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structuraldraEgene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.
Expression of AniA, the Major Anaerobically Induced Outer Membrane Protein of
Neisseria gonorrhoeae
, Provides Protection against Killing by Normal Human Sera
Authors: Cardinale J. A. , Clark V. L.
Published: July 2002

Anaerobically grown
Neisseria gonorrhoeae
has previously been shown to have elevated serum resistance in the absence of exogenous CMP-
N
-acetylneuraminic acid or detectable sialylation. We hypothesized that the anaerobically induced gonococcal outer membrane protein AniA might have a role in this phenomenon, as it is the only known gonococcal protein that is absent under aerobic conditions. An
N. gonorrhoeae
F62 derivative, RUG7035, in which
aniA
is under control of the
tac
promoter, was used to examine the effect of AniA expression on serum resistance. In this study, we found that expression of AniA enhanced the serum resistance of
N. gonorrhoeae
and may account for these earlier observations.
CD4+Depletion Selectively Inhibits Eosinophil Recruitment to the Cornea and AbrogatesOnchocerca volvulusKeratitis (River Blindness)
Authors: Hall Laurie R. , Kaifi Jussuf T. , Diaconu Eugenia , Pearlman Eric
Published: July 2002

Previous studies demonstrated that in the murine model ofOnchocerca volvuluskeratitis, neutrophils and eosinophils are recruited into the cornea in a biphasic manner in response to intrastromal injection. To determine if CD4+T cells regulate migration of neutrophils and eosinophils into the cornea, CD4+cells were depleted using monoclonal antibody GK1.5 before intrastromal injection of parasite antigens. Depletion of CD4+cells abrogated corneal opacification at later but not early stages of disease. Consistent with this observation, CD4 depletion significantly impaired recruitment of eosinophils to the cornea but had no effect on neutrophils. These data indicate that CD4+T cells mediate sustainedO. volvuluskeratitis by regulating eosinophil recruitment to the cornea.
High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A
Authors: Pless Dorothy D. , Torres Edna R. , Reinke Emily K. , Bavari Sina
Published: July 2002

Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H
C
. Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H
C
was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A H
C
fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.
Generation and Surface Localization of Intact M Protein inStreptococcus pyogenesAre Dependent onsagA
Authors: Biswas Indranil , Germon Pierre , McDade Kathleen , Scott June R.
Published: July 2002

The M protein is an important surface-located virulence factor ofStreptococcuspyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that thesaglocus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription ofemm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation insagA, the first gene in thesaglocus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of theemmgene was unaffected by thesagAmutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in thesagAmutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made thesagAmutant strain susceptible to phagocytosis. Thus, althoughsagAdoes not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor.
Codon Optimization of Gene Fragments Encoding
Plasmodium falciparum
Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice
Authors: Narum David L. , Kumar Sanjai , Rogers William O. , Fuhrmann Steven R. , Liang Hong , Oakley Miranda , Taye Alem , Sim B. Kim Lee , Hoffman Stephen L.
Published: July 2002

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of
Plasmodium falciparum
gene sequences (∼80%) is much greater than that of
Homo sapiens
(∼59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of
P. falciparum
genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased
P. falciparum
protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa
P. falciparum
erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the
P. falciparum
merozoite surface protein 1 (MSP-1
42
) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 μg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.
Virulence ofLeishmania infantumIs Expressed as a Clonal and Dominant Phenotype in Experimental Infections
Authors: Garin Yves Jean-François , Sulahian Annie , Pratlong Francine , Meneceur Pascale , Gangneux Jean-Pierre , Prina Eric , Dedet Jean-Pierre , Derouin Francis
Published: July 2002

HumanLeishmania infantuminfection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence inL. infantumvisceral leishmaniasis is clonal and dominant in nature.
Differential Changes in Heat Shock Protein-, Lipoarabinomannan-, and Purified Protein Derivative-Specific Immunoglobulin G1 and G2 Isotype Responses during BovineMycobacterium aviumsubsp.paratuberculosisInfection
Authors: Koets Ad P. , Rutten Victor P. M. G. , de Boer Masja , Bakker Douwe , Valentin-Weigand Peter , van Eden Willem
Published: July 2002

Bovine paratuberculosis is caused by infection of young calves withMycobacterium aviumsubsp.paratuberculosis. In some of the chronically infected cows the long asymptomatic stage (2 to 4 years) is followed by a rapid progression to a clinical stage due to protein-losing enteropathy, which will ultimately be fatal. The current dogma is that in early stages of disease the cell-mediated responses predominate, whereas in the clinical stage of the disease the humoral responses prevail, possibly signaling a switch in immune reactivity related to disease progression. We developed immunoglobulin M (IgM)-, IgA-, and IgG1- and IgG2-isotype-specific enzyme-linked immunosorbent assays forM. aviumsubsp.paratuberculosis-derived antigens (heat shock proteins of 70 kDa [Hsp70] and 65 kDa [Hsp65], lipoarabinomannan [LAM], andM. aviumsubsp.paratuberculosispurified protein derivative PPD [PPDP]). The serological responses of cows in different stages of paratuberculosis were used to evaluate the putative shift in immune responsiveness. In the clinical stage the PPDP-specific IgG1 responses were increased compared to those in the asymptomatic stage. However, total IgG1 and IgG2 and the Hsp70-, Hsp65-, and LAM-specific isotype responses were decreased in the clinical stage were decreased compared to those in the asymptomatic stage of disease. Thus, the classical pattern was found only for PPDP antigens and the IgG1 isotype. For other antigens and isotypes and the total IgG levels, the response pattern is different and indicates that there is no uniform association with increased antibody responses during the progression from the asymptomatic stage to the clinical stage of bovine paratuberculosis.
Expression of C-Reactive Protein in the Human Respiratory Tract
Authors: Gould Jane M. , Weiser Jeffrey N.
Published: July 2002

C-reactive protein (CRP) is a normal constituent of human sera synthesized by hepatocytes and induced by proinflammatory cytokines. The function of this acute-phase reactant includes activation of complement and enhancement of opsonophagocytosis. CRP binds to phosphorylcholine (ChoP), a constituent of eukaryotic membranes that is also found on the cell surface of major bacterial pathogens of the human respiratory tract, includingStreptococcus pneumoniaeandHaemophilus influenzae. The presence of CRP on mucosal surfaces and role in innate immunity in the human respiratory tract where ChoP-containing organisms reside have not been previously studied. We have shown using a monoclonal antibody to CRP that CRP is present in inflamed (0.17 to 42 μg/ml) and uninflamed (<0.05 to 0.88 μg/ml) secretions from the human respiratory tract in sufficient quantities for an antimicrobial effect. In addition, the CRP gene was expressed in epithelial cells of the human respiratory tract using in situ hybridization on nasal polyps and reverse transcriptase PCR of pharyngeal cells in culture. The complement-dependent bactericidal activity of normal nasal airway surface fluid and sputum against ChoP-expressingH. influenzaewas abolished when the secretions were pretreated to remove CRP. In summary, the results indicate that CRP is present in secretions of the human respiratory tract, that human respiratory epithelial cells are capable of CRP expression, and that this protein may contribute to bacterial clearance in the human respiratory tract.
Upregulation of Toll-Like Receptor 2 Gene Expression in Macrophage Response to Peptidoglycan and High Concentration of Lipopolysaccharide Is Involved in NF-κB Activation
Authors: Liu Yan , Wang Yin , Yamakuchi Munekazu , Isowaki Sumikazu , Nagata Etsuro , Kanmura Yuichi , Kitajima Isao , Maruyama Ikuro
Published: July 2002

Toll-like receptors 2 and 4 (TLR2 and TLR4) have been found to transduce signals of peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively, for NF-κB activation. However, little is known about the expression and regulation of the TLR2 gene in monocytes/macrophages in response to the two typical bacterial products. We show in the present study that both PGN and a high concentration of LPS increase TLR2 gene expression in macrophage-like cells, 1α,25-dihydroxyvitamin D3-differentiated human HL60 and mouse RAW264.7 cells, and human monocytes in a dose- and time-dependent manner. Actinomycin D and pyrrolidine dithiocarbamate inhibition of gene transcription and NF-κB activation, respectively, blocks LPS- and PGN-elevated TLR2 mRNA in monocytic cells. The LPS-induced increase in TLR2 mRNA in monocytic cells is abolished by polymyxin B pretreatment and is observed in peripheral blood mononuclear cells from pigs subjected to endotoxic shock. Further, high concentrations of LPS and synthetic lipid A increase TLR2 mRNA expression in peritoneal macrophages from both TLR4-deficient C3H/HeJ mice and normal C3H/HeN mice, a process that constitutes induction of TLR4-independent TLR2 expression. These findings demonstrate that TLR2 gene expression is upregulated in macrophage responses to PGN and to high concentrations of LPS in vitro and in vivo and correlates with NF-κB activation.
CD4+T Helper 1 Cells Facilitate Regression of Murine Lyme Carditis
Authors: Bockenstedt Linda K. , Kang Insoo , Chang Christopher , Persing David , Hayday Adrian , Barthold Stephen W.
Published: July 2002

Murine Lyme borreliosis, caused by infection with the spirocheteBorrelia burgdorferi, results in acute arthritis and carditis that regress as a result ofB. burgdorferi-specific immune responses.B. burgdorferi-specific antibodies can attenuate arthritis in mice deficient in both B cells and T cells but have no effect on carditis. Because macrophages comprise the principal immune cell in carditis, T-cell responses that augment cell-mediated immunity may be important for carditis regression. To investigate this hypothesis, we examined the course of Lyme carditis in mice selectively deficient in B cells or αβ T cells. Our results show that carditis regresses in B-cell-deficient B10.Akmice but not in αβ T-cell-deficient mice, independently of the mouse strain background. Despite prominent macrophage infiltrates, hearts fromB. burgdorferi-infected αβ T-cell-deficient mice had less mRNA for tumor necrosis factor alpha as measured by reverse transcription-PCR compared to infected control mice. Anti-inflammatory cytokine mRNA levels were equivalent. Adoptive transfer of gamma interferon-secreting CD4+T cells into infected αβ T-cell-deficient mice promoted carditis resolution. These results show that αβ T cells can promote resolution of murine Lyme carditis and are the first demonstration of a beneficial role for CD4+T helper 1 cells in this disease.
Cloning, Sequencing, and Expression of the Leukotoxin Gene fromFusobacterium necrophorum
Authors: Narayanan Sanjeev Kumar , Nagaraja T. G. , Chengappa M. M. , Stewart George C.
Published: July 2002

Fusobacterium necrophorumis a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF;lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the wholelktAORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin fromF. necrophorumculture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed inEscherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against theF. necrophorumleukotoxin. Southern blot hybridization analysis revealed different patterns oflktAhybridizing bands between isolates of the two subspecies ofF. necrophorum.
DNA Vaccine Combinations Expressing Either Tissue Plasminogen Activator Signal Sequence Fusion Proteins or Ubiquitin-Conjugated Antigens Induce Sustained Protective Immunity in a Mouse Model of Pulmonary Tuberculosis
Authors: Delogu Giovanni , Li Amy , Repique Charlene , Collins Frank , Morris Sheldon L.
Published: July 2002

DNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis. In this study, we evaluated the effectiveness of immunizing with combinations of 10 different tuberculosis DNA vaccines that expressed mycobacterial proteins fused at the N terminus to eukaryotic intracellular targeting sequences. In one vaccine combination, the genes were fused to the tissue plasminogen activator signal sequence (TPA), while in a second combination the same 10 genes were expressed as ubiquitin (Ub)-conjugated proteins. In ex vivo studies in which the secretion of gamma interferon was measured, cellular immune responses were detected in mice vaccinated with either the TPA DNA vaccine combination or the Ub DNA vaccine combination at 7 and 14 days following a low-doseMycobacterium tuberculosischallenge. Moreover, mice vaccinated with the TPA combination, the Ub combination, andMycobacterium bovisBCG were able to limit the growth of tubercle bacilli in the lung and spleen after a virulent tuberculous aerosol challenge. Histopathological analyses also showed that mice immunized with the DNA vaccine combinations had substantially improved postinfection lung pathology relative to the naïve controls. Finally, in three different long-term experiments, the survival periods following aerogenic challenge were extended as much as sevenfold for vaccinated mice compared to naïve controls. Interestingly, in all three experiments, no significant differences were detected in the mean times to death for mice immunized with the TPA combination or the Ub combination relative to the BCG controls. In conclusion, these studies demonstrate the effectiveness of immunization with DNA vaccine combinations against tuberculosis and suggest that further testing of these plasmid cocktails is warranted.
Failure of theMycobacterium bovisBCG Vaccine: Some Species of Environmental Mycobacteria Block Multiplication of BCG and Induction of Protective Immunity to Tuberculosis
Authors: Brandt Lise , Feino Cunha Joana , Weinreich Olsen Anja , Chilima Ben , Hirsch Penny , Appelberg Rui , Andersen Peter
Published: July 2002

The efficacy ofMycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of theMycobacterium aviumcomplex were found to block BCG activity completely.
Penicillium marneffeiCauses Osteopontin-Mediated Production of Interleukin-12 by Peripheral Blood Mononuclear Cells
Authors: Koguchi Yoshinobu , Kawakami Kazuyoshi , Kon Shigeyuki , Segawa Tatsuya , Maeda Masahiro , Uede Toshimitsu , Saito Atsushi
Published: July 2002

We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated withPenicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured withP. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14+cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion fromP. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the β-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation withP. marneffeiand that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN byP. marneffei-stimulated PBMCs.
Safety and Immunogenicity of a Prototype EnterotoxigenicEscherichia coliVaccine Administered Transcutaneously
Authors: Güereña-Burgueño Fernando , Hall Eric R. , Taylor David N. , Cassels Frederick J. , Scott Daniel A. , Wolf Marcia K. , Roberts Zachary J. , Nesterova Galina V. , Alving Carl R. , Glenn Gregory M.
Published: July 2002

Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenicEscherichia coli(ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.
TheBabesia bovisMerozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins
Authors: Florin-Christensen Monica , Suarez Carlos E. , Hines Stephen A. , Palmer Guy H. , Brown Wendy C. , McElwain Terry F.
Published: July 2002

Members of the variable merozoite surface antigen (vmsa) gene family ofBabesia bovisencode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containingmsa-2, a member of thevmsafamily, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies ofmsa-2-related genes were found in the locus. The four genes, designatedmsa-2a1(which corresponds to the originally describedmsa-2gene),msa-2a2,msa-2b, andmsa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a1) amino acids. MSA-2a1and -2a2are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a2and -2b. In contrast,msa-2cis most closely related to the previously describedbabr 0.8gene in Australia strains ofB. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain ofB. boviswith the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a1, -2a2, -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a1, -2b, and -2c. The results support the evolution of themsa-2locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.
Inhibition of Leukocyte Rolling by Nitric Oxide during Sepsis Leads to Reduced Migration of Active Microbicidal Neutrophils
Authors: Benjamim Claudia Farias , Silva João Santana , Fortes Zuleica Bruno , Oliveira Maria Aparecida , Ferreira Sérgio Henrique , Cunha Fernando Queiroz
Published: July 2002

We developed two models of sepsis with different degrees of severity, sublethal and lethal sepsis, induced by cecal ligation and puncture. Lethal sepsis induced by cecal ligation and puncture (L-CLP) resulted in failure of neutrophil migration to the infection site and high mortality. Treatment of septic animals with aminoguanidine (AG), a nitric oxide (NO) synthase inhibitor, precluded the failure of neutrophil migration and protected the animals from death. However, cytokine-induced NO synthase (iNOS)-deficient (iNOS−/−) mice subjected to L-CLP did not present neutrophil migration failure, but 100% lethality occurred. iNOS−/−mice subjected to sublethal sepsis induced by cecal ligation and puncture (SL-CLP) also suffered high mortality despite the occurrence of neutrophil migration. This apparent paradox could be explained by the lack of microbicidal activity in neutrophils of iNOS−/−mice present at the infection site due to their inability to produce NO. Notably, SL- and L-CLP iNOS−/−mice showed high bacterial numbers in exudates. The inhibition of neutrophil migration by NO is due to inhibition of a neutrophil/endothelium adhesion mechanism, since a reduction in leukocyte rolling, adhesion, and emigration was observed in L-CLP wild-type mice. These responses were prevented by AG treatment and were not observed in the iNOS−/−L-CLP group. There was no significant change in L-selectin expression in neutrophils from L-CLP mice. Thus, it seems that the decrease in leukocyte rolling is due to a defect in the expression of adhesion molecules on endothelial surfaces mediated by iNOS-derived NO. In conclusion, the results indicate that despite the importance of NO in neutrophil microbicidal activity, its generation in severe sepsis reduces neutrophil migration by inhibiting leukocyte rolling and their firm adhesion to the endothelium, in effect impairing the migration of leukocytes and consequently their fundamental role in host cell defense mechanisms.
Candida albicans
Expresses a Focal Adhesion Kinase-Like Protein That Undergoes Increased Tyrosine Phosphorylation upon Yeast Cell Adhesion to Vitronectin and the EA.hy 926 Human Endothelial Cell Line
Authors: Santoni Giorgio , Lucciarini Roberta , Amantini Consuelo , Jacobelli Jordan , Spreghini Elisabetta , Ballarini Patrizia , Piccoli Mario , Gismondi Angela
Published: July 2002

The signaling pathways triggered by adherence of
Candida albicans
to the host cells or extracellular matrix are poorly understood. We provide here evidence in
C. albicans
yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain
C. albicans
yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited
C. albicans
yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of αvβ3 and αvβ5 integrin-like on
C. albicans
either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in
C. albicans
yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a
C. albicans
mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that
C. albicans
Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.
Enzymology of Type IV Macromolecule Secretion Systems: the Conjugative Transfer Regions of Plasmids RP4 and R388 and the
cag
Pathogenicity Island of
Helicobacter pylori
Encode Structurally and Functionally Related Nucleoside Triphosphate Hydrolases
Authors: Krause Sabine , Pansegrau Werner , Lurz Rudi , de la Cruz Fernando , Lanka Erich
Published: July 2002

Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the
cag
pathogenicity island of
Helicobacter pylori
(HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.
Branched-Chain α-Keto Acid Catabolism via the Gene Products of the
bkd
Operon in
Enterococcus faecalis
: a New, Secreted Metabolite Serving as a Temporary Redox Sink
Authors: Ward Donald E. , van der Weijden Coen C. , van der Merwe Marthinus J. , Westerhoff Hans V. , Claiborne Al , Snoep Jacky L.
Published: July 2002

Recently the
bkd
gene cluster from
Enterococcus faecalis
was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain α-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the
bkd
gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the
bkd
gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain α-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the α-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of α-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD
+
from the NADH produced by the branched-chain α-keto acid dehydrogenase complex was required for complete conversion of α-ketoisocaproate. Interestingly, during the conversion of the branched-chain α-keto acids an intermediate was always detected extracellularly. With α-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1,1-dihydroxy-4-methyl-2-pentanone. This reduced form of α-ketoisocaproic acid was found to serve as a temporary redox sink.
Regulation of Cation-Coupled High-Affinity Phosphate Uptake in the Yeast
Saccharomyces cerevisiae
Authors: Pattison-Granberg Johanna , Persson Bengt L.
Published: July 2002

Studies of the high-affinity phosphate transporters in the yeast
Saccharomyces cerevisiae
using mutant strains lacking either the Pho84 or the Pho89 permease revealed that the transporters are differentially regulated. Although both genes are induced by phosphate starvation, activation of the Pho89 transporter precedes that of the Pho84 transporter early in the growth phase in a way which may possibly reflect a fine tuning of the phosphate uptake process relative to the availability of external phosphate.
ZipA-Induced Bundling of FtsZ Polymers Mediated by an Interaction between C-Terminal Domains
Authors: Hale Cynthia A. , Rhee Amy C. , de Boer Piet A. J.
Published: July 2002

FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division inEscherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA−cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.