bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Response Kinetics of Gamma Interferon and Interleukin-4 in Bovine Tuberculosis
Authors: Rhodes S. G. , Palmer N. , Graham S. P. , Bianco A. E. , Hewinson R. G. , Vordermeier H. M.
Published: July 2002

This study shows that gamma interferon (IFN-γ) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected withMycobacterium bovis. The different kinetics of the IFN-γ and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection withM. bovisare described. An early increase in IFN-γ was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose ofM. boviswhich did not result in the development of lesions, positive DTH skin test, or substantial IFN-γ responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infectedM. bovisfield reactors showed increased IFN-γ and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of theM. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematodeOnchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.
Absence of a Cysteine Protease Effect on Bacterial Virulence in Two Murine Models of Human Invasive Group A Streptococcal Infection
Authors: Ashbaugh Cameron D. , Wessels Michael R.
Published: July 2002

The cysteine protease of group A streptococci has been suggested to contribute to the pathogenesis of invasive infection through degradation of host tissue, activation of the host inflammatory response, release of protective molecules from the bacterial cell surface, or other mechanisms. However, studies of the effects on virulence of inactivating the cysteine protease gene
speB
have yielded conflicting results. In some reports, a
speB
mutant was relatively avirulent in mouse models of invasive infection whereas little or no attenuation of virulence was observed in other studies of similar mutant strains. Possible reasons for these discordant results include differences in the streptococcal strains from which the
speB
mutants were derived, differences in the infection models employed, or unintended effects on another virulence determinant(s) that arose during the derivation of a
speB
mutant. We attempted to clarify these issues by characterizing the phenotypic properties and relative virulence in mice of two
speB
mutant strains, both derived from wild-type strain AM3:
speB
mutant AM3speB, which has been shown to be markedly attenuated in virulence in mice after intraperitoneal or subcutaneous challenge, and AM3speBΩ, a new mutant strain derived for this investigation. Both mutant strains were negative for protease activity, as expected, and both produced wild-type amounts of type 3 M protein and streptolysin O. However, AM3speB produced significantly less cell-associated hyaluronic acid capsule than did parent strain AM3 or strain AM3speBΩ. Compared to wild-type strain AM3, AM3speB was more sensitive to opsonophagocytic killing in vitro and was significantly less virulent in mice after intraperitoneal challenge. By contrast, AM3speBΩ was fully resistant to phagocytosis and did not differ significantly from the wild-type strain in mouse virulence after an intraperitoneal or subcutaneous challenge. We concluded that previous reports attributing loss of virulence in strain AM3speB to inactivation of
speB
are in error. Within the limitations of the models used, we found no effect of cysteine protease on invasive streptococcal infection.
Comparative Analysis of Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Activity in Serum and Lethality in Mice and Rabbits Pretreated with the Staphylococcal Superantigen Toxic Shock Syndrome Toxin 1
Authors: Dinges Martin M. , Schlievert Patrick M.
Published: July 2002

Host susceptibility to lipopolysaccharide (LPS) is correlated with the levels of circulating tumor necrosis factor alpha (TNF-α) that develop in response to circulating LPS. Mice are resistant, relative to rabbits, to the lethal effects of LPS. This study indicates that mice and rabbits are equally sensitive to the lethal effects of circulating TNF-α but that mice are more resistant than rabbits to the induction of circulating TNF-α by LPS.
Brucella abortus
Genes Identified following Constitutive Growth and Macrophage Infection
Authors: Eskra Linda , Canavessi Aurea , Carey Merriann , Splitter Gary
Published: July 2002

The chronicity of
Brucella abortus
infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for
Brucella
, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for
Brucella
. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced
B. abortus
genes. Constitutively producing
gfp
clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively produced
gfp
in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K
+
transport gene), and site-specific recombination (
xerD
gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5′ phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of
B. abortus
genes that are activated following macrophage invasion provides insight into
Brucella
pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified
Brucella
genes.
Mice Lacking Interleukin-2 (IL-2)/IL-15 Receptor β Chain Are Susceptible to Infection with AvirulentSalmonella entericasubsp.entericaSerovar Choleraesuis but Mice Lacking IL-2 Are Resistant
Authors: Nishimura Hitoshi , Tagaya Mitsuhiko , Tsunobuchi Hironaka , Suzuki Haruhiko , Nakashima Izumi , Yoshikai Yasunobu
Published: July 2002

Interleukin-2 (IL-2)/IL-15 receptor β (IL-15Rβ)−/−mice were susceptible to infection with avirulentSalmonella entericasubsp.entericaserovar Choleraesuis, whereas IL-2−/−mice were resistant. A natural killer cell response was not evident for both types of deficient mice. A Th1 response was detected in IL-2−/−but not in IL-2/IL-15Rβ−/−mice infected withSalmonella, suggesting that IL-2/IL-15Rβ signaling is important for the generation of protective Th1 cells.
Enhanced Immunogenicity of CD4+T-Cell Responses and Protective Efficacy of a DNA-Modified Vaccinia Virus Ankara Prime-Boost Vaccination Regimen for Murine Tuberculosis
Authors: McShane Helen , Brookes Roger , Gilbert Sarah C. , Hill Adrian V. S.
Published: July 2002

DNA vaccines whose DNA encodes a variety of antigens fromMycobacterium tuberculosishave been evaluated for immunogenicity and protective efficacy. CD8+T-cell responses and protection achieved in other infectious disease models have been optimized by using a DNA immunization to prime the immune system and a recombinant virus encoding the same antigen(s) to boost the response. A DNA vaccine (D) and recombinant modified vaccinia virus Ankara (M) in which the DNA encodes early secreted antigenic target 6 and mycobacterial protein tuberculosis 63 synthesized, and each was found to generate specific gamma interferon (IFN-γ)-secreting CD4+T cells. Enhanced CD4+IFN-γ T-cell responses were produced by both D-M and M-D immunization regimens. Significantly higher levels of IFN-γ were seen with a D-D-D-M immunization regimen. The most immunogenic regimens were assessed in a challenge study and found to produce protection equivalent to that produced byMycobacterium bovisBCG. Thus, heterologous prime-boost regimens boost CD4+as well as CD8+T-cell responses, and the use of heterologous constructs encoding the same antigen(s) may improve the immunogenicity and protective efficacy of DNA vaccines against tuberculosis and other diseases.
Antigenic Variation of
Anaplasma Marginale
: Major Surface Protein 2 Diversity during Cyclic Transmission between Ticks and Cattle
Authors: Barbet A. F. , Yi Jooyoung , Lundgren Anna , McEwen B. R. , Blouin E. F. , Kocan K. M.
Published: July 2002

The rickettsial pathogen
Anaplasma marginale
expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of
A. marginale
and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of
A. marginale
infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain
A. marginale
transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each
A. marginale
population. The major sequence variants did not change on passage of
A. marginale
between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.
Plasmodium falciparum
Homologue of the Genes for
Plasmodium vivax
and
Plasmodium yoelii
Adhesive Proteins, Which Is Transcribed but Not Translated
Authors: Taylor Helen M. , Triglia Tony , Thompson Jenny , Sajid Mohammed , Fowler Ruth , Wickham Mark E. , Cowman Alan F. , Holder Anthony A.
Published: July 2002

The 235-kDa family of rhoptry proteins in
Plasmodium yoelii
and the two reticulocyte binding proteins of
P. vivax
comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in
P. falciparum
(
PfRH3
) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5′ end of the gene.
gp96-Peptide Vaccination of Mice against Intracellular Bacteria
Authors: Zügel Ulrich , Sponaas Anne-Marit , Neckermann Jutta , Schoel Bernd , Kaufmann Stefan H. E.
Published: July 2002

This work demonstrates that gp96 preparations isolated from cells infected with intracellular bacteria induce cytotoxic T-lymphocyte responses and confer protection. Our findings extend previous reports on the immunogenicity of gp96-associated peptides to antigens derived from intracellular bacteria. Immunization with gp96 may therefore represent a promising vaccination strategy against bacterial pathogens.
Interleukin-6 Enhances Production of Anti-OspC Immunoglobulin G2b Borreliacidal Antibody
Authors: Remington Monica C. , Munson Erik L. , Callister Steven M. , Molitor Melanie L. , Christopherson John A. , DeCoster David J. , Lovrich Steven D. , Schell Ronald F.
Published: July 2002

Protection against infection withBorrelia burgdorferiis dependent primarily on induction of complement-dependent antibody that can kill the spirochete. Measuring the production of sustained high levels of borreliacidal antibody is thus paramount for determining potential vaccine efficacy. We investigated the borreliacidal antibody response in sera and the amount of antibody produced by cultured lymph node cells of C3H/HeJ mice vaccinated with outer surface protein C (OspC). We showed that recombinant OspC was a weak stimulant of borreliacidal antibody production compared to whole cells of OspC-expressingB. burgdorferi. Mice vaccinated withB. burgdorferiin adjuvant produced a high level (titer, 5,120) of anti-OspC borreliacidal antibody, which waned rapidly. Similarly, borreliacidal antibody production by cultured lymph node cells from vaccinated mice peaked soon after vaccination and then decreased. Treatment of lymph node cells with interleukin-6 (IL-6) augmented borreliacidal antibody production, particularly immunoglobulin G2b, whereas treatment with anti-IL-6 inhibited the borreliacidal response. These findings demonstrate a previously unrecognized role for IL-6 in borreliacidal antibody production that may have important implications for vaccine development.
Characterization of a Stress-Induced Alternate Sigma Factor, RpoS, of
Coxiella burnetii
and Its Expression during the Development Cycle
Authors: Seshadri Rekha , Samuel James E.
Published: July 2002

Coxiella burnetii
is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment.
C. burnetii
has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of
C. burnetii
. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an
rpoD
homologue, and the stress response sigma factor, encoded by an
rpoS
homologue. The
rpoS
gene was cloned by complementation of an
Escherichia coli rpoS
null mutant containing an RpoS-dependent
lacZ
fusion (
osmY
::
lacZ
). Expression of
C. burnetii rpoS
was regulated by growth phase in
E. coli
(induced upon entry into stationary phase). A glutathione
S
-transferase–RpoS fusion protein was used to develop polyclonal antiserum against
C. burnetii
RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment.
RecombinantActinobacillus actinomycetemcomitansCytolethal Distending Toxin Proteins Are Required To Interact To Inhibit Human Cell Cycle Progression and To Stimulate Human Leukocyte Cytokine Synthesis
Authors: Akifusa Sumio , Poole Stephen , Lewthwaite Jo , Henderson Brian , Nair Sean P.
Published: July 2002

It has recently been discovered thatActinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene fromA. actinomycetemcomitansinEscherichia coliand purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1β (IL-1β), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.
Plasmodium knowlesiProvides a Rapid In Vitro and In Vivo Transfection System That Enables Double-Crossover Gene Knockout Studies
Authors: Kocken Clemens H. M. , Ozwara Hastings , van der Wel Annemarie , Beetsma Annette L. , Mwenda Jason M. , Thomas Alan W.
Published: July 2002

Transfection technology for malaria parasites provides a valuable tool for analyzing gene function and correlating genotype with phenotype. Transfection models are even more valuable when appropriate animal models are available in addition to complete in vitro systems to be able to fully analyze parasite-host interactions. Here we describe the development of such a model by using the nonhuman primate malariaPlasmodium knowlesi. Blood-stage parasites were adapted to long-term in vitro culture. In vitro-adapted parasites could readapt to in vivo growth and regain wild-type characteristics after a single passage through an intact rhesus monkey.P. knowlesiparasites, either in vitro adapted or in vivo derived, were successfully transfected to generate circumsporozoite protein (CSP) knockout parasites by double-crossover mechanisms. In vitro-transfected and cloned CSP knockout parasites were derived in a time span of only 18 days. Microscopic evaluation of developing oocysts from mosquitoes that had fed on CSP knockout parasites confirmed the impairment of sporozoite formation observed inP. bergheiCSP knockout parasites. TheP. knowlesimodel currently is the only malaria system that combines rapid and precise double-crossover genetic manipulation procedures with complete in vitro as well as in vivo possibilities. This allows for full analysis ofP. knowlesigenotype-phenotype relationships and host-parasite interactions in a system closely related to humans.
Functional Analysis of theHelicobacter pylori cagPathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms
Authors: Selbach Matthias , Moese Stefan , Meyer Thomas F. , Backert Steffen
Published: July 2002

The type IV secretion machinery encoded by thecagpathogenicity island (PAI) ofHelicobacter pylorihas been implicated in a series of host responses during infection. Here, we analyzed the function of 12cagPAI genes from bothcagI andcagII loci, including the completevirB/Dcomplex (virB4, virB7, virB8, virB9, virB10, virB11, andvirD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering (“hummingbird”) phenotype uponH. pyloriinfection of AGS gastric epithelial cells. For the first time, we have complemented individualcagPAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on allvirB/Dgenes and several other genes of thecagPAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.
Deletion of the Two-Component Histidine Kinase Gene (
CHK1
) of
Candida albicans
Contributes to Enhanced Growth Inhibition and Killing by Human Neutrophils In Vitro
Authors: Torosantucci Antonella , Chiani Paola , De Bernardis Flavia , Cassone Antonio , Calera Jose Antonio , Calderone Richard
Published: July 2002

We have observed that human neutrophils (polymorphonuclear leukocytes [PMNs]) have an increased growth-inhibitory and killing effect on a strain of
Candida albicans
with a deletion of
CHK1
, a gene encoding a putative histidine kinase. The PMN effect was not due to increased phagocytosis of the null strain. This observation may partially explain the reduced virulence in a hematogenously disseminated murine model of candidiasis.
Site-Specific Proteolysis of the MALP-404 Lipoprotein Determines the Release of a Soluble Selective Lipoprotein-Associated Motif-Containing Fragment and Alteration of the Surface Phenotype of
Mycoplasma fermentans
Authors: Davis Kelley L. , Wise Kim S.
Published: July 2002

The mature MALP-404 surface lipoprotein of
Mycoplasma fermentans
comprises a membrane-anchored N-terminal lipid-modified region responsible for macrophage activation (P. F. Mühlradt, M. Kieβ, H. Meyer, R. Süβmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997) and an external hydrophilic region that contains the selective lipoprotein-associated (SLA) motif defining a family of lipoproteins from diverse but selective prokaryotes, including mycoplasmas (M. J. Calcutt, M. F. Kim, A. B. Karpas, P. F. Mühlradt, and K. S. Wise, Infect. Immun. 67:760-771, 1999). This family generally corresponds to a computationally defined group of orthologs containing the basic membrane protein (BMP) domain. Two discrete lipid-modified forms of the abundant MALP product which vary dramatically in ratio among isolates of
M. fermentans
occur on the mycoplasma surface: (i) MALP-404, the full-length mature product, and (ii) MALP-2, the Toll-like receptor 2-mediated macrophage-activating lipopeptide containing the N-terminal 14 residues of the mature lipoprotein. The role of posttranslational processing in the biogenesis of MALP-2 from the prototype MALP-404 SLA-containing lipoprotein was investigated. Detergent phase fractionation of cell-bound products and N-terminal sequencing of a newly discovered released fragment (RF) demonstrated that MALP-404 was subject to site-specific proteolysis between residues 14 and 15 of the mature lipoprotein, resulting in the cell-bound MALP-2 and soluble RF products. This previously unknown mechanism of posttranslational processing among mycoplasmas suggests that specific cleavage of some surface proteins may confer efficient “secretion” of extracellular products by these organisms, with concurrent changes in the surface phenotype. This newly identified form of variation may have significant implications for host adaptation by mycoplasmas, as well as other pathogens expressing lipoproteins of the SLA (BMP) family.
Protective Immunity againstMycobacterium tuberculosisInduced by Dendritic Cells Pulsed with both CD8+- and CD4+-T-Cell Epitopes from Antigen 85A
Authors: McShane Helen , Behboudi Shahriar , Goonetilleke Nilu , Brookes Roger , Hill Adrian V. S.
Published: July 2002

Immunization with DNA followed by modified vaccinia virus Ankara strain, both expressing the antigen 85A, induced both CD4+- and CD8+-T-cell responses in BALB/c mice. Following challenge withMycobacterium tuberculosis, this prime-boost regimen produced protection equivalent to that conferred byMycobacterium bovisBCG. Following immunization with dendritic cells pulsed with an antigen 85A CD4+- or CD8+-restricted epitope, alone or in combination, copresentation of both epitopes on the same dendritic cell was required for protection, demonstrating that induced CD8+T cells can play a protective role against tuberculosis.
Carcinoembryonic Antigen Family Receptor Recognition by Gonococcal Opa Proteins Requires Distinct Combinations of Hypervariable Opa Protein Domains
Authors: Bos Martine P. , Kao David , Hogan Daniel M. , Grant Christopher C. R. , Belland Robert J.
Published: July 2002

Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.
Local Role for Tumor Necrosis Factor Alpha in the Pulmonary Inflammatory Response toMycobacterium tuberculosisInfection
Authors: Smith Sherilyn , Liggitt Denny , Jeromsky Elizabeth , Tan Xiaoxia , Skerrett Shawn J. , Wilson Christopher B.
Published: July 2002

The local intrapulmonary role of tumor necrosis factor alpha (TNF-α) in a protective host response during acute and chronic infection withMycobacterium tuberculosisis incompletely understood. To directly assess its role in the intrapulmonary immune response, we compared the responses of transgenic mice with a local pulmonary blockade of TNF-α (SPCTNFRIIFc mice) to mice with globally inhibited TNF-α (TNFRKO mice) and mice with normal immune systems (control mice). Consistent with previous reports, 100% of TNFRKO mice died by 28 days after aerosol infection, and these mice had markedly increased numbers of bacteria and widespread tissue necrosis in their lungs compared to controls. The median survival time of the SPCTNFRIIFc mice was 142 days, and 75% died by 180 days. Even though the numbers of bacteria in the lungs of the SPCTNFRIIFc mice were marginally increased compared to controls, these mice had a persistent neutrophilic inflammatory response and increased expression of proinflammatory cytokines (interleukin-1α/β [IL-1α/β], IL-18, gamma interferon, IL-6, and macrophage migration inhibitory factor) and chemokines (eotaxin, macrophage inflammatory protein 1α/β, gamma interferon-inducible protein 10, macrophage chemotaxic protein 1, and TCA-3) in their lungs. These studies with the SPCTNFRIIFc mice provide direct evidence for the local importance of TNF-α in the proper regulation of host defense toM. tuberculosis. The studies also suggest that when the local actions of TNF-α are selectively impaired in the lungs, tissue destruction and death ensue, at least in part, due to persistent expression of proinflammatory mediators that would normally be downregulated.
Genome-Based Identification of Chromosomal Regions Specific for
Salmonella
spp
Authors: Hansen-Wester Imke , Hensel Michael
Published: July 2002

Acquisition of genomic elements by horizontal gene transfer represents an important mechanism in the evolution of bacterial species. Pathogenicity islands are a subset of horizontally acquired elements present in various pathogens. These elements are frequently located adjacent to tRNA genes. We performed a comparative genome analysis of
Salmonella enterica
serovars Typhi and Typhimurium and
Escherichia coli
and scanned tRNA loci for the presence of species-specific, horizontally acquired genomic elements. A large number of species-specific elements were identified. Here, we describe the characteristics of four large chromosomal insertions at tRNA genes of
Salmonella
spp. The tRNA-associated elements harbor various genes previously identified as single virulence genes, indicating that these genes have been acquired with large chromosomal insertions. Southern blot analyses confirmed that the tRNA-associated elements are specific to
Salmonella
and also indicated a heterogeneous distribution within the salmonellae. Systematic scanning for insertions at tRNA genes thus represents a tool for the identification of novel pathogenicity islands.
Construction, Characterization, and Animal Testing of WRSd1, a
Shigella dysenteriae
1 Vaccine
Authors: Venkatesan Malabi M. , Hartman Antoinette B. , Newland John W. , Ivanova Vessela S. , Hale Thomas L. , McDonough Marie , Butterton Joan
Published: July 2002

WRSd1 is a
Shigella dysenteriae
1 vaccine containing deletions of the
virG(icsA)
gene required for intercellular spreading and a 20-kb chromosomal region encompassing the Shiga toxin genes (
stxAB
). WRSd1 was constructed from
S. dysenteriae
1 strain 1617 that was originally isolated during the 1968 to 1969 epidemic of Shiga dysentery in Guatemala. The
virG(icsA)
deletion was constructed from a streptomycin-resistant (Str
r
) mutant of 1617 by a filter mating procedures using a
virG(icsA)
deletion derivative, pΔvirG2. A colony that was invasive for HeLa cells and negative for the
virG(icsA)
gene by Southern blotting was grown anaerobically on plates containing chlorate for selection of resistant colonies that had lost the entire Shiga toxin gene. A
virG(icsA) stxAB
Str
r
mutant selected from the chlorate plates was designated WRSd1. This candidate vaccine was evaluated for safety, immunogenicity, and protective efficacy using the guinea pig keratoconjunctivitis model. WRSd1 was Sereny negative, and two applications of this strain to the cornea elicited a significant protective immune response against the
S. dysenteriae
1 O antigen. Vaccination with WRSd1 conferred protection against challenge with each of three virulent
S. dysenteriae
1 strains. Since a vaccine protecting against multiple
Shigella
species is required for most areas where
Shigella
is endemic, protection studies using a combination vaccine of
Shigella sonnei
vaccine strain WRSS1,
Shigella flexneri
2a vaccine strain SC602, and WRSd1 were also performed. Guinea pigs vaccinated with a mixture of equal amounts of the three vaccine strains were protected against challenge with each of the homologous virulent strains. Unlike WRSS1 and SC602, however, the level of protection afforded by WRSd1 in a combination vaccine was lower than the protection elicited by a pure culture. A current Good Manufacturing Practice product of WRSd1 given intragastrically to rhesus monkeys proved safe and immunogenic.
Gene Rearrangements in the
vsa
Locus of
Mycoplasma pulmonis
Authors: Shen Xuejun , Gumulak Juliann , Yu Huilan , French C. Todd , Zou Nianxiang , Dybvig Kevin
Published: July 2002

The
vsa
genes of
Mycoplasma pulmonis
encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six
vsa
genes comprising a 10-kb region of the genome of
M. pulmonis
strain KD735-15. In the current study,
vsa
-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11
vsa
genes. The middle region of the
vsa
locus contains a large open reading frame (ORF) that is not a
vsa
gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine
vsa
genes in a strain of
M. pulmonis
that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15
vsaA
, -
C
, -
E
, and -
F
genes and two unique genes (
vsaG
and
vsaH
) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of
vsa
recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the
vrs
box is proposed.
Another Unusual Type of Citric Acid Cycle Enzyme in
Helicobacter pylori
: the Malate:Quinone Oxidoreductase
Authors: Kather Birgit , Stingl Kerstin , van der Rest Michel E. , Altendorf Karlheinz , Molenaar Douwe
Published: July 2002

The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the
Helicobacter pylori
genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of
H. pylori
. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in
mqo
deletion mutants of
Escherichia coli
or
Corynebacterium glutamicum
. Furthermore, this plasmid was able to complement the phenotype of the
C. glutamicum mqo
deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that
H. pylori
possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.
Rho-Dependent Transcription Termination in the
tna
Operon of
Escherichia coli
: Roles of the
boxA
Sequence and the
rut
Site
Authors: Konan Kouacou Vincent , Yanofsky Charles
Published: July 2002

Expression of the tryptophanase (
tna
) operon of
Escherichia coli
is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region,
tnaC
, containing a single, crucial Trp codon. Studies with a
lacZ
reporter construct lacking the
tnaC-tnaA
spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in
cis
on the ribosome translating
tnaC
to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the
boxA
sequence and the
rut
site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of
boxA
or introducing specific point mutations in
boxA
results in high-level constitutive expression. Some constitutive changes introduced in
boxA
do not change the TnaC peptide sequence. We confirm that deletion of the
rut
site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the
tnaC
start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the
tnaC
coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a
boxA
point mutation with
rut
site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating
tnaC
blocks Rho's access to the
boxA
and
rut
sites, thereby preventing transcription termination.
Two Active Forms of UDP-
N
-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria
Authors: Du Wensheng , Brown James R. , Sylvester Daniel R. , Huang Jianzhong , Chalker Alison F. , So Chi Y. , Holmes David J. , Payne David J. , Wallis Nicola G.
Published: July 2002

Gene sequences encoding the enzymes UDP-
N
-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one
murA
gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two
murA
genes of the gram-positive organism
Streptococcus pneumoniae
were studied further. Each of the
murA
genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its
murA
genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-
N
-acetylglucosamine, confirming they are both active UDP-
N
-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.
Oxazolidinone Resistance Mutations in 23S rRNA of
Escherichia coli
Reveal the Central Region of Domain V as the Primary Site of Drug Action
Authors: Xiong Liqun , Kloss Patricia , Douthwaite Stephen , Andersen Niels Møller , Swaney Steven , Shinabarger Dean L. , Mankin Alexander S.
Published: July 2002

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected
Escherichia coli
oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.
Novel Formaldehyde-Activating Enzyme in
Methylobacterium extorquens
AM1 Required for Growth on Methanol
Authors: Vorholt Julia A. , Marx Christopher J. , Lidstrom Mary E. , Thauer Rudolf K.
Published: July 2002

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the α-proteobacterium
Methylobacterium extorquens
AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from
M. extorquens
AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO
2
. Mutants of
M. extorquens
AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.
Cysteine-Scanning Mutagenesis of the Periplasmic Loop Regions of PomA, a Putative Channel Component of the Sodium-Driven Flagellar Motor in
Vibrio alginolyticus
Authors: Asai Yukako , Shoji Tomokazu , Kawagishi Ikuro , Homma Michio
Published: July 2002

The sodium-driven motor consists of the products of at least four genes,
pomA
,
pomB
,
motX
, and
motY
, in
Vibrio alginolyticus
. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop
1-2
) and at positions 167 to 180 between membrane segments 3 and 4 (loop
3-4
). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop
3-4
impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and
N
-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop
3-4
. None of the loop
1-2
residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.
High-Resolution Physical Map of the
Sinorhizobium meliloti
1021 pSyma Megaplasmid
Authors: Barloy-Hubler Frederique , Capela Delphine , Barnett Melanie J. , Kalman Sue , Federspiel Nancy A. , Long Sharon R. , Galibert Francis
Published: July 2002

To facilitate sequencing of the
Sinorhizobium meliloti
1021 pSyma megaplasmid, a high-resolution map was constructed by ordering 113 overlapping bacterial artificial chromosome clones with 192 markers. The 157 anonymous sequence tagged site markers (81,072 bases) reveal hypothetical functions encoded by the replicon.
Multiple Gene Products and Sequences Required for Excision of the Mobilizable Integrated
Bacteroides
Element NBU1
Authors: Shoemaker Nadja B. , Wang Gui-Rong , Salyers Abigail A.
Published: July 2002

NBU1 is an integrated 10.3-kbp
Bacteroides
element, which can excise and transfer to
Bacteroides
or
Escherichia coli
recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene,
intN1
, was necessary and sufficient for integration of NBU1 into its target site on the
Bacteroides
or
E. coli
genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to
intN1
, four open reading frames plus a large region downstream of the fourth gene,
prmN1
. This downstream sequence was designated XRS, for “excision-required sequence.” XRS contains the
oriT
of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene,
mobN1
. This is the first time an
oriT
, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of
intN1
,
orf2
, completely abolished excision. The next open reading frame,
orf2x
, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of
orf2x
,
orf3
and
prmN1
. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying
prmN1
and part of the XRS (XRS
HIII
) inhibited excision when provided in
trans
on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying
prmN1
, XRS, and the entire mobilization gene,
mobN1
, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a
cis
-acting region (XRS) which includes the
oriT
. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the
oriT
during conjugal transfer, to prevent premature nicking at the
oriT
before NBU1 has excised and circularized.
KorSA from the
Streptomyces
Integrative Element pSAM2 Is a Central Transcriptional Repressor: Target Genes and Binding Sites
Authors: Sezonov Guennadi , Possoz Christophe , Friedmann Annick , Pernodet Jean-Luc , Guérineau Michel
Published: July 2002

pSAM2, a 10.9-kb mobile integrative genetic element from
Streptomyces ambofaciens
, possesses, as do a majority of
Streptomyces
conjugative plasmids, a
kil-kor
system associated with its transfer. The
kor
function of pSAM2 was attributed to the
korSA
gene, but its direct role remained unclear. The present study was focused on the determination of the KorSA targets. It was shown that KorSA acts as a transcriptional repressor by binding to a conserved 17-nucleotide sequence found upstream of only two genes: its own gene,
korSA
, and
pra
, a gene positively controlling pSAM2 replication, integration, and excision. A unique feature of KorSA, compared to Kor proteins from other
Streptomyces
conjugative plasmids, is that it does not directly regulate pSAM2 transfer. KorSA does not bind to the pSAM2 genes coding for transfer and intramycelial spreading. Through the repression of
pra
, KorSA is able to negatively regulate pSAM2 functions activated by Pra and, consequently, to maintain pSAM2 integrated in the chromosome.