bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucosal Immune Responses and Protection against Tetanus Toxin after Intranasal Immunization with RecombinantLactobacillus plantarum
Authors: Grangette Corinne , Müller-Alouf Heide , Goudercourt Denise , Geoffroy Marie-Claude , Turneer Mireille , Mercenier Annick
Published: July 2002

The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, aLactobacillus plantarumstrain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.
Novel Genetic and Phenotypic Heterogeneity in
Bordetella bronchiseptica
Pertactin
Authors: Register Karen B.
Published: July 2002

The
Bordetella bronchiseptica
outer membrane protein pertactin is believed to function as an adhesin and is an important protective immunogen. Previous sequence analysis of the pertactin gene identified two regions predicted to encode amino acid repeat motifs. Recent studies have documented DNA sequence heterogeneity in both regions. The present study describes additional variants in these regions, which form the basis for six novel pertactin types. Immunoblotting demonstrated phenotypic heterogeneity in pertactin consistent with the predicted combined sizes of the repeat regions. A revised system for classifying
B. bronchiseptica
pertactin variants is proposed.
Induction of Inducible Nitric Oxide Synthase-NO· by Lipoarabinomannan ofMycobacterium tuberculosisIs Mediated by MEK1-ERK, MKK7-JNK, and NF-κB Signaling Pathways
Authors: Chan Edward D. , Morris Kristin R. , Belisle John T. , Hill Preston , Remigio Linda K. , Brennan Patrick J. , Riches David W. H.
Published: July 2002

Nitric oxide (NO· ) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism againstMycobacterium tuberculosisin mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways in the regulation of iNOS and NO· by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-κB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-γ)-ManLAM-induced iNOS protein and NO2−expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-γ–ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IκBα, a potent inhibitor of NF-κB activation, confirmed that the IκBα kinase (IKK)–NF-κB signaling pathway enhanced IFN-γ–ManLAM-induced iNOS promoter activity. By contrast, activated p38mapkinhibited iNOS induction. These results indicate that combined IFN-γ and ManLAM stimulation induced iNOS and NO· expression and that MEK1-ERK, MKK7-JNK, IKK–NF-κB, and p38mapksignaling pathways play important regulatory roles.
Increased Immunogenicity and Induction of Class Switching by Conjugation of Complement C3d to Pneumococcal Serotype 14 Capsular Polysaccharide
Authors: Test Samuel T. , Mitsuyoshi Joyce , Connolly Charles C. , Lucas Alexander H.
Published: July 2002

Previous studies have demonstrated an adjuvant effect for the C3d fragment of complement C3 when coupled to T-dependent protein antigens. In this study, we examined the antibody response to covalent conjugates of C3d and a T-independent antigen, the capsular polysaccharide of serotype 14Streptococcus pneumoniae(PPS14). We prepared a conjugate of mouse C3d and PPS14 and compared its immunogenicity with that of a conjugate of PPS14 and ovalbumin (OVA). When BALB/c mice were immunized with PPS14-C3d, there was a significant increase in serum anti-PPS14 concentrations compared with either native PPS14 or control PPS14-glycine conjugates. This was accompanied by a switch in anti-PPS14 from predominantly immunoglobulin M (IgM) to IgG1 by day 25 following primary immunization. Following secondary immunization with PPS14-C3d, there was a marked booster response and a further increase in the ratio of IgG1 to IgM anti-PPS14. Although the primary antibody response to the PPS14-OVA conjugate exceeded that induced by immunization with PPS14-C3d, serum anti-PPS14 concentrations after a second injection of PPS14-C3d were nearly identical to those induced by secondary immunization with PPS14-OVA. Experiments with athymic nude mice suggested that T cells were not required for the adjuvant effect of C3d on the primary immune response to PPS14 but were necessary for enhancement of the memory response after a second injection of PPS14-C3d. These studies show that the adjuvant effects of C3d extend to T-independent antigens as well as T-dependent antigens. As a means of harnessing the adjuvant potential of the innate immune system, C3d conjugates may prove useful as a component of vaccines against encapsulated bacteria.
Down-Regulation of Glycosylphosphatidylinositol-Specific Phospholipase D Induced by Lipopolysaccharide and Oxidative Stress in the Murine Monocyte- Macrophage Cell Line RAW 264.7
Authors: Du Xiaohan , Low Martin G.
Published: July 2002

Serum glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) activity is reduced over 75% in systemic inflammatory response syndrome. To investigate the mechanism of this response, expression of the GPI-PLD gene was studied in the mouse monocyte-macrophage cell line RAW 264.7 stimulated with lipopolysaccharide (LPS; 0.5 to 50 ng/ml). GPI-PLD mRNA was reduced approximately 60% in a time- and dose-dependent manner. Oxidative stress induced by 0.5 mM H2O2or 50 μM menadione also caused a greater than 50% reduction in GPI-PLD mRNA. The antioxidantN-acetyl-l-cysteine attenuated the down-regulatory effect of H2O2but not of LPS. Cotreatment of the cells with actinomycin D inhibited down-regulation induced by either LPS or H2O2. The half-life of GPI-PLD mRNA was not affected by LPS, or decreased slightly with H2O2, indicating that the reduction in GPI-PLD mRNA is due primarily to transcriptional regulation. Stimulation with tumor necrosis factor alpha (TNF-α) resulted in ∼40% reduction in GPI-PLD mRNA in human A549 alveolar carcinoma cells but not RAW 264.7 cells, suggesting that alternative pathways could exist in different cell types for down-regulating GPI-PLD expression during an inflammatory response and the TNF-α autocrine signaling mechanism alone is not sufficient to recapitulate the LPS-induced reduction of GPI-PLD in macrophages. Sublines of RAW 264.7 cells with reduced GPI-PLD expression exhibited increased cell sensitivity to LPS stimulation and membrane-anchored CD14 expression on the cell surface. Our data suggest that down-regulation of GPI-PLD could play an important role in the control of proinflammatory responses.
Complete DNA Sequence and Analysis of the Large Virulence Plasmid of
Shigella flexneri
Authors: Venkatesan Malabi M. , Goldberg Marcia B. , Rose Debra J. , Grotbeck Erik J. , Burland Valerie , Blattner Frederick R.
Published: July 2002

The complete sequence analysis of the 210-kb
Shigella flexneri
5a virulence plasmid was determined.
Shigella
spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known
Shigella
virulence determinants are encoded on a large plasmid that is unique to virulent strains of
Shigella
and enteroinvasive
Escherichia coli
; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of
shet2
and five alleles of
ipaH
were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new
Shigella
virulence determinants.
Periodontal Pathogens Produce Quorum Sensing Signal Molecules
Authors: Frias Jorge , Olle Ester , Alsina Merce
Published: July 2002

Different species of bacteria important in the composition of dental plaque were tested for production of extracellular autoinducer-like activities that stimulate the expression of the luminescence genes in
Vibrio harveyi
. Several strains of
Prevotella intermedia, Fusobacterium nucleatum
, and
Porphyromonas gingivalis
were found to produce such activities. Interestingly, these bacteria belong to the same phylogenetic group, and they are periodontal pathogens important in the development of periodontal disease. They specifically produce extracellular signaling molecule related autoinducer-2 from
V. haveyi
. Nevertheless, they seem to be unable to produce homologues of acyl-homoserine lactones. Furthermore,
Escherichia coli
DH5α can be complemented by the introduction of a
P. gingivalis
gene with high homology to the
luxS
gene, which has been called
luxS
P.g.
.
Biological Activity of a C-Terminal Fragment ofPasteurella multocidaToxin
Authors: Busch Christian , Orth Joachim , Djouder Nabil , Aktories Klaus
Published: July 2002

The protein toxin ofPasteurella multocidaPMT is a potent mitogen and activator of phospholipase Cβ. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.
Diepitopic Construct of Functionally and Epitopically Complementary Peptides Enhances Immunogenicity, Reactivity with Glucosyltransferase, and Protection from Dental Caries
Authors: Taubman Martin A. , Holmberg Cynthia J. , Smith Daniel J.
Published: July 2002

Coimmunization with peptide constructs from catalytic (CAT) and glucan-binding (GLU) domains of glucosyltransferase (GTF) of mutans streptococci has resulted in enhanced levels of antibody to the CAT construct and to GTF. We designed and synthesized a diepitopic construct (CAT-GLU) containing two copies of both CAT (B epitope only) and GLU (B and T epitope) peptides. The immunogenicity of this diepitopic construct was compared with that of individual CAT and GLU constructs by immunizing groups of Sprague-Dawley rats subcutaneously in the salivary gland vicinity with the CAT-GLU, CAT, or GLU construct or by treating rats by sham immunization. Levels of serum immunoglobulin G (IgG) antibody to GTF or CAT in the CAT-GLU group were significantly greater than in GLU- or CAT-immunized groups. Immunization with CAT-GLU was compared to coimmunization with a mixture of CAT and GLU in a second rodent experiment under a similar protocol. CAT-GLU immunization resulted in serum IgG and salivary IgA responses to GTF and CAT which were greater than after coimmunization. Immunization with the diepitopic construct and communization with CAT and GLU constructs showed proliferation of T lymphocytes to GTF. Immunization with either the CAT or GLU construct has been shown to elicit significant protection in a rodent dental caries model. Similarly in this study, the enhanced response to GTF after immunization with the CAT-GLU construct resulted in protective effects on dental caries. Therefore, the CAT-GLU diepitopic construct can be a potentially important antigen for a caries vaccine, giving rise to greater immune response than after immunization with CAT, GLU, or a mixture of the two.
Traversal of
Candida albicans
across Human Blood-Brain Barrier In Vitro
Authors: Jong Ambrose Y. , Stins Monique F. , Huang Sheng-He , Chen Steven H. M. , Kim Kwang Sik
Published: July 2002

Candida albicans
is an opportunistic pathogen, which primarily affects neonates and immunocompromised individuals. The pathogen can invade the central nervous system, resulting in meningitis. At present, the pathogenesis of
C. albicans
meningitis is unclear. We used an in vitro model of the human blood-brain barrier to investigate the interaction(s) of
C. albicans
with human brain microvascular endothelial cells (BMEC). Binding of
C. albicans
to human BMEC was time and inoculum dependent. Invasion of
C. albicans
into human BMEC was demonstrated by using an enzyme-linked immunosorbent assay based on fluorescent staining of
C. albicans
with calcoflour. In contrast, avirulent
Candida
mutant strains and nonpathogenic yeast
Saccharomyces cerevisiae
were not able to bind and invade human BMEC. Morphological studies revealed that on association with human BMEC,
C. albicans
formed germ tubes and was able to bud intracellularly. Transmission electron microscopy showed various stages of
C. albicans
interactions with human BMEC, e.g., pseudopod-like structures on human BMEC membrane and intracellular vacuole-like structures retaining
C. albicans
. Of interest,
C. albicans
was able to bud and develop pseudohyphae inside human BMEC without apparent morphological changes of the host cells. In addition,
C. albicans
penetrates through human BMEC monolayers without a detectable change in transendothelial electrical resistance and inulin permeability. This is the first demonstration that
C. albicans
is able to adhere, invade, and transcytose across human BMEC without affecting monolayer integrity. A complete understanding of the interaction(s) of
C. albicans
with human BMEC should contribute to the understanding of the pathogenic mechanism(s) of
C. albicans
meningitis.
Role of Inflammatory Mediators in Resistance and Susceptibility to Pneumococcal Infection
Authors: Kerr Alison R. , Irvine June J. , Search Jennifer J. , Gingles Neill A. , Kadioglu Aras , Andrew Peter W. , McPheat William L. , Booth Charles G. , Mitchell Tim. J.
Published: July 2002

Variations in the host response during pneumonia caused byStreptococcus pneumoniaein susceptible (CBA/Ca) and resistant (BALB/c) inbred mouse strains were investigated. Significant differences were detected in survival time, core body temperature, lung-associated and systemic bacterial loads, mast cell numbers, magnitude and location of cytokine production, lung disruption, and ability of isolated lung cells to release the cytokine tumor necrosis factor (TNF) alpha in vitro. Overall, the results indicate that the reduced capacity of CBA/Ca mice to induce rapid TNF activity within the airways following infection withS. pneumoniaemay be a factor in their elevated susceptibility to pneumococcal pneumonia.
A Gene from the Locus of Enterocyte Effacement That Is Required for EnteropathogenicEscherichia coliTo Increase Tight-Junction Permeability Encodes a Chaperone for EspF
Authors: Elliott Simon J. , O'Connell Colin B. , Koutsouris Athanasia , Brinkley Carl , Donnenberg Michael S. , Hecht Gail , Kaper James B.
Published: July 2002

Disruption of the barrier properties of the enterocyte tight junction is believed to be important in the pathogenesis of diarrhea caused by enteropathogenicEscherichia coli(EPEC). This phenotype can be measured in vitro as the ability of EPEC to reduce transepithelial resistance (TER) across enterocyte monolayers and requires the products of the locus of enterocyte effacement (LEE) and, in particular, the type III secreted effector protein EspF. We report a second LEE-encoded gene that is also necessary for EPEC to fully reduce TER.rorf10is not necessary for EPEC adherence, EspADB secretion, or formation of attaching and effacing lesions. However,rorf10mutants have a diminished TER phenotype, reduced intracellular levels of EspF, and a reduced ability to translocate EspF into epithelial cells. The product ofrorf10is a 14-kDa intracellular protein rich in α-helices that specifically interacts with EspF but not with Tir or other EPEC secreted proteins. These properties are consistent with the hypothesis thatrorf10encodes a type III secretion chaperone for EspF, and we rename this protein CesF, the chaperone for EPEC secreted protein F.
DsbA and DsbC Are Required for Secretion of Pertussis Toxin by
Bordetella pertussis
Authors: Stenson Trevor H. , Weiss Alison A.
Published: July 2002

The Dsb family of enzymes catalyzes disulfide bond formation in the gram-negative periplasm, which is required for folding and assembly of many secreted proteins. Pertussis toxin is arguably the most complex toxin known: it is assembled from six subunits encoded by five genes (for subunits S1 to S5), with 11 intramolecular disulfide bonds. To examine the role of the Dsb enzymes in assembly and secretion of pertussis toxin, we identified and mutated the
Bordetella pertussis dsbA
,
dsbB
, and
dsbC
homologues. Mutations in
dsbA
or
dsbB
resulted in decreased levels of S1 (the A subunit) and S2 (a B-subunit protein), demonstrating that DsbA and DsbB are required for toxin assembly. Mutations in
dsbC
did not impair assembly of periplasmic toxin but resulted in decreased toxin secretion, suggesting a defect in the formation of the Ptl secretion complex.
Construction and Characterization of an Acapsular Mutant of
Mannheimia haemolytica
A1
Authors: McKerral Linda J. , Lo Reggie Y. C.
Published: July 2002

The
nmaA
and
nmaB
genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in
Mannheimia haemolytica
A1. A chloramphenicol resistance (Cm
r
) cassette cloned behind an
M. haemolytica
A1 promoter,
plpcat
, was created and used to interrupt
nmaA
and
nmaB
. A 1.3-kbp DNA fragment that encompasses part of
nmaA
and
nmaB
was replaced by the 1.0-kbp
plpcat
, resulting in a knockout mutant which is Cm
r
and unable to synthesize
N
-acetylmannosamine (ManNAc) and
N
-acetylmannosaminuronic acid (ManNAcA). The DNA replacement was confirmed by Southern hybridization and PCR analyses of the
nmaA
and
nmaB
loci. Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain. The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum. Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.
Effect of Low- and High-Virulence
Yersinia enterocolitica
Strains on the Inflammatory Response of Human Umbilical Vein Endothelial Cells
Authors: Denecker Geertrui , Tötemeyer Sabine , Mota Luís J. , Troisfontaines Paul , Lambermont Isabelle , Youta Carine , Stainier Isabelle , Ackermann Martin , Cornelis Guy R.
Published: July 2002

Pathogenic strains of
Yersinia
spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different
Yersinia enterocolitica
strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B)
Y. enterocolitica
strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence
Y. enterocolitica
strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun.
69:
7652-7662, 2001) all the
Y. enterocolitica
strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.
Comparative Phenotypic Analysis of theBordetella parapertussisIsolate Chosen for Genomic Sequencing
Authors: Heininger Ulrich , Cotter Peggy A. , Fescemyer Howard W. , Martinez de Tejada Guillermo , Yuk Ming H. , Miller Jeff F. , Harvill Eric T.
Published: July 2002

The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain ofBordetella parapertussischosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains ofB. pertussis(Tohama I),B. bronchiseptica(RB50), and other isolates ofB. parapertussis. Multiple in vitro and in vivo assays distinguished each species.B. parapertussiswas more similar toB. bronchisepticathan toB. pertussisin many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetellaantibodies, and serum antimicrobial resistance. In other assays,B. parapertussiswas distinct from all other species (in pigment production) or more similar toB. pertussis(by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.
In Vivo Effects of a Synthetic 2-Kilodalton Macrophage-Activating Lipopeptide ofMycoplasma fermentansafter Pulmonary Application
Authors: Lührmann Anke , Deiters Ursula , Skokowa Julia , Hanke Michaela , Gessner Johannes E. , Mühlradt Peter F. , Pabst Reinhard , Tschernig Thomas
Published: July 2002

Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals. Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes. The present study deals with the question of which mycoplasmal components cause this host reaction. The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection. To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2. Application of 2.5 μg induced a pronounced leukocyte accumulation in the bronchoalveolar space. At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3. Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation. The leukocyte count of the lung interstitium was increased on day 3 after treatment. After 10 days all investigated cell populations returned to control levels. Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates. MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2. Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction. The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.
Progressive Bovine Paratuberculosis Is Associated with Local Loss of CD4+T Cells, Increased Frequency of γδ T Cells, and Related Changes in T-Cell Function
Authors: Koets Ad , Rutten Victor , Hoek Aad , van Mil Frans , Müller Kerstin , Bakker Douwe , Gruys Erik , van Eden Willem
Published: July 2002

Bovine paratuberculosis is caused by the infection of young calves withMycobacterium aviumsubsp.paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4+T-cell frequency and a significant increase in TcR1-N12+γδ T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4+T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4+T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.
Characterization of the Fructosyltransferase Gene of
Actinomyces naeslundii
WVU45
Authors: Bergeron Lori J. , Morou-Bermudez Evangelia , Burne Robert A.
Published: July 2002

Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of
d
-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated from
Actinomyces naeslundii
WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5′ from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving
ftf
expression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimal
ftf
expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that
ftf
was constitutively transcribed. Thus, the
A. naeslundii
FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.
Decoupling of Genome Size and Sequence Divergence in a Symbiotic Bacterium
Authors: Wernegreen Jennifer J. , Ochman Howard , Jones Isaac B. , Moran Nancy A.
Published: July 2002

In contrast to genome size variation in most bacterial taxa, the small genome size of
Buchnera
sp. was shown to be highly conserved across genetically diverse isolates (630 to 643 kb). This exceptional size conservation may reflect the inability of this obligate mutualist to acquire foreign DNA and reduced selection for genetic novelty within a static intracellular environment.
SecB Dependence of an Exported Protein Is a Continuum Influenced by the Characteristics of the Signal Peptide or Early Mature Region
Authors: Kim Jinoh , Luirink Joen , Kendall Debra A.
Published: July 2002

We have used
Escherichia coli
alkaline phosphatase to show the interplay among the characteristics of two amino-terminal domains in the preprotein (the signal peptide and the early mature region), the efficiency with which this protein is transported, and its requirement for SecB to accomplish the transport process. The results suggest that although alkaline phosphatase does not normally require SecB for transport, it is inherently able to utilize SecB, and it does so when its ability to interface with the transport machinery is compromised.
Identification of a Second Region of the Spo0A Response Regulator of
Bacillus subtilis
Required for Transcription Activation
Authors: Rowe-Magnus Dean A. , Richer Martin J. , Spiegelman George B.
Published: July 2002

Deletion of the 10 C-terminal amino acids of the
Bacillus subtilis
response regulator Spo0A or valine substitution at D258 and L260 resulted in a sporulation-negative phenotype and loss of in vivo activation of the
spoIIG
and
spoIIA
operon promoters. Repression of the
abrB
promoter was not affected by the mutations. In combination with the previously characterized mutation (A257V), the results identify amino acids at positions 257, 258, and 260 as being required for transcription activation by Spo0A.
Autoregulation of the
Pseudomonas aeruginosa
Protein PtxS Occurs through a Specific Operator Site within the
ptxS
Upstream Region
Authors: Swanson Britta L. , Hamood Abdul N.
Published: July 2002

We have previously shown that the
Pseudomonas aeruginosa toxA
regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3′ end of the 52-bp fragment is located 58 bp 5′ of the
ptxS
translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates
ptxS
expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and
ptxS
expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the
ptxS
promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in
ptxS
expression in the
P. aeruginosa
strain PAO1 and the
ptxS
isogenic mutant PAO::
ptxS
. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in
ptxS
expression in both PAO1 and PAO::
ptxS
. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate
ptxS
expression.
Gene Expression Analysis of the
Streptococcus pneumoniae
Competence Regulons by Use of DNA Microarrays
Authors: Peterson Scott , Cline Robin T. , Tettelin Hervé , Sharov Vasily , Morrison Donald A.
Published: July 2002

Competence for genetic transformation in
Streptococcus pneumoniae
is coordinated by the competence-stimulating peptide (CSP), which induces a sudden and transient appearance of competence during exponential growth in vitro. Models of this quorum-sensing mechanism have proposed sequential expression of several regulatory genes followed by induction of target genes encoding DNA-processing-pathway proteins. Although many genes required for transformation are known to be expressed only in response to CSP, the relative timing of their expression has not been established. Overlapping expression patterns for the genes
cinA
and
comD
(G. Alloing, B. Martin, C. Granadel, and J. P. Claverys, Mol. Microbiol. 29:75–83, 1998) suggest that at least two distinct regulatory mechanisms may underlie the competence cycle. DNA microarrays were used to estimate mRNA levels for all known competence operons during induction of competence by CSP. The known competence regulatory operons,
comAB
,
comCDE
, and
comX
, exhibited a low or zero initial (uninduced) signal, strongly increased expression during the period between 5 and 12 min after CSP addition, and a decrease nearly to original values by 15 min after initiation of exposure to CSP. The remaining competence genes displayed a similar expression pattern, but with an additional delay of approximately 5 min. In a mutant defective in ComX, which may act as an alternate sigma factor to allow expression of the target competence genes, the same regulatory genes were induced, but the other competence genes were not. Finally, examination of the expression of 60 candidate sites not previously associated with competence identified eight additional loci that could be induced by CSP.
The Level of
DAL80
Expression Down-Regulates GATA Factor-Mediated Transcription in
Saccharomyces cerevisiae
Authors: Cunningham Thomas S. , Rai Rajendra , Cooper Terrance G.
Published: July 2002

Nitrogen-catabolic gene expression in
Saccharomyces cerevisiae
is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors. In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the
GAT1
,
DAL80
, and
DEH1
genes all contain multiple GATA sequences as well. These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation. Here we show, using
DAL80
fused to the carbon-regulated
GAL1
,
10
or copper-regulated
CUP1
promoter, that
GAT1
expression is inversely regulated by the level of
DAL80
expression, i.e., as
DAL80
expression increases,
GAT1
expression decreases. The amount of
DAL80
expression also dictates the level at which
DAL3
, a gene activated almost exclusively by Gln3p, is transcribed. Gat1p was found to partially substitute for Gln3p in transcription. These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled. Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.
A Gene Cluster Involved in Metal Homeostasis in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: García-Domínguez Mario , Lopez-Maury Luis , Florencio Francisco J. , Reyes José C.
Published: July 2002

A gene cluster composed of nine open reading frames (ORFs) involved in Ni
2+
, Co
2+
, and Zn
2+
sensing and tolerance in the cyanobacterium
Synechocystis
sp. strain PCC 6803 has been identified. The cluster includes an Ni
2+
response operon and a Co
2+
response system, as well as a Zn
2+
response system previously described. Expression of the Ni
2+
response operon (
nrs
) was induced in the presence of Ni
2+
and Co
2+
. Reduced Ni
2+
tolerance was observed following disruption of two ORFs of the operon (
nrsA
and
nrsD
). We also show that the
nrsD
gene encodes a putative Ni
2+
permease whose carboxy-terminal region is a metal binding domain. The Co
2+
response system is composed of two divergently transcribed genes,
corR
and
corT
, mutants of which showed decreased Co
2+
tolerance. Additionally,
corR
mutants showed an absence of Co
2+
-dependent induction of
corT
, indicating that CorR is a transcriptional activator of
corT
. To our knowledge, CorR is the first Co
2+
-sensing transcription factor described. Our data suggest that this region of the
Synechocystis
sp. strain PCC 6803 genome is involved in sensing and homeostasis of Ni
2+
, Co
2+
, and Zn
2+
.
Dual Roles of
Bradyrhizobium japonicum
Nickelin Protein in Nickel Storage and GTP-Dependent Ni Mobilization
Authors: Olson Jonathan W. , Maier Robert J.
Published: July 2002

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N
2
-fixing, H
2
-oxidizing bacterium
Bradyrhizobium japonicum
. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in
B. japonicum
, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.
A C-Terminal Disulfide Bridge in Pediocin-Like Bacteriocins Renders Bacteriocin Activity Less Temperature Dependent and Is a Major Determinant of the Antimicrobial Spectrum
Authors: Fimland Gunnar , Johnsen Line , Axelsson Lars , Brurberg May B. , Nes Ingolf F. , Eijsink Vincent G. H. , Nissen-Meyer Jon
Published: July 2002

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37°C, whereas wild-type sakacin P was approximately 10 times less potent at 37°C than at 20°C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys→Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.
Expression of UGA-Containing
Mycoplasma
Genes in
Bacillus subtilis
Authors: Kannan T. R. , Baseman Joel B.
Published: July 2002

We used
Bacillus subtilis
to express UGA-containing
Mycoplasma
genes encoding the P30 adhesin (one UGA) of
Mycoplasma pneumoniae
and methionine sulfoxide reductase (two UGAs) of
Mycoplasma genitalium
. Due to natural UGA suppression, these
Mycoplasma
genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type
Bacillus
. The
B. subtilis
-expressed
Mycoplasma
proteins appeared as single bands and not as multiple bands compared to expression in recombinant
Escherichia coli. Bacillus
mutants carrying mutations in the structural gene (
prfB
) for release factor 2 markedly enhanced the level of readthrough of UGA-containing
Mycoplasma
genes.
Rubrerythrin and Rubredoxin Oxidoreductase inDesulfovibrio vulgaris: a Novel Oxidative Stress Protection System
Authors: Lumppio Heather L. , Shenvi Neeta V. , Summers Anne O. , Voordouw Gerrit , Kurtz Donald M.
Published: July 2002

Evidence is presented for an alternative to the superoxide dismutase (SOD)-catalase oxidative stress defense system inDesulfovibrio vulgaris(strain Hildenborough). This alternative system consists of the nonheme iron proteins, rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo), the product of therbogene (also called desulfoferrodoxin). A Δrbostrain ofD. vulgariswas found to be more sensitive to internal superoxide exposure than was the wild type. Unlike Rbo, expression of plasmid-borne Rbr failed to restore the aerobic growth of a SOD-deficient strain ofEscherichia coli. Conversely, plasmid-borne expression of two different Rbrs fromD. vulgarisincreased the viability of a catalase-deficient strain ofE. colithat had been exposed to hydrogen peroxide whereas Rbo actually decreased the viability. A previously undescribedD. vulgarisgene was found to encode a protein having 50% sequence identity to that ofE. coliFe-SOD. This gene also encoded an extended N-terminal sequence with high homologies to export signal peptides of periplasmic redox proteins. The SOD activity ofD. vulgarisis not affected by the absence of Rbo and is concentrated in the periplasmic fraction of cell extracts. These results are consistent with a superoxide reductase rather than SOD activity of Rbo and with a peroxidase activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic oxidative stress protection system inD. vulgarisand other anaerobic microorganisms is proposed.
β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in
Streptomyces coelicolor
A3(2)
Authors: Revill W. Peter , Bibb Maureen J. , Scheu Ann-Karolin , Kieser Helen J. , Hopwood David A.
Published: July 2002

The
Streptomyces coelicolor fab
(fatty acid biosynthesis) gene cluster (
fabD-fabH-acpP-fabF
) is cotranscribed to produce a leaderless mRNA transcript. One of these genes,
fabH
, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in
S. coelicolor
.
Virulence Plasmid-Borne
spvB
and
spvC
Genes Can Replace the 90-Kilobase Plasmid in Conferring Virulence to
Salmonella enterica
Serovar Typhimurium in Subcutaneously Inoculated Mice
Authors: Matsui Hidenori , Bacot Christopher M. , Garlington Wendy A. , Doyle Thomas J. , Roberts Steve , Gulig Paul A.
Published: July 2002

In a mouse model of systemic infection, the
spv
genes carried on the
Salmonella enterica
serovar Typhimurium virulence plasmid increase the replication rate of salmonellae in host cells of the reticuloendothelial system, most likely within macrophages. A nonpolar deletion in the
spvB
gene greatly decreased virulence but could not be complemented by
spvB
alone. However, a low-copy-number plasmid expressing
spvBC
from a constitutive
lacUV5
promoter did complement the
spvB
deletion. By examining a series of
spv
mutations and cloned
spv
sequences, we deduced that
spvB
and
spvC
could be sufficient to confer plasmid-mediated virulence to
S. enterica
serovar Typhimurium. The
spvBC
-bearing plasmid was capable of replacing all of the
spv
genes, as well as the entire virulence plasmid, of serovar Typhimurium for causing systemic infection in BALB/c mice after subcutaneous, but not oral, inoculation. A point mutation in the
spvBC
plasmid preventing translation but not transcription of
spvC
eliminated the ability of the plasmid to confer virulence. Therefore, it appears that both
spvB
and
spvC
encode the principal effector factors for Spv- and plasmid-mediated virulence of serovar Typhimurium.
Two-Component System That Regulates Methanol and Formaldehyde Oxidation in
Paracoccus denitrificans
Authors: Harms N. , Reijnders W. N. M. , Koning S. , van Spanning R. J. M.
Published: July 2002

A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from
Paracoccus denitrificans
. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (
fhlA
) was undetectable in the mutant, and expression of the
S
-formylglutathione hydrolase gene (
fghA
) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C
1
metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.