bluuebunny/crossref_metadata_2025 huggingface dataset:
An
rpsL
Cassette, Janus, for Gene Replacement through Negative Selection in
Streptococcus pneumoniae
Authors: Sung C. K. , Li H. , Claverys J. P. , Morrison D. A.
Published: July 2002

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of
Streptococcus pneumoniae
. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (
kan
) and a counterselectable
rpsL
+
marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in
S. pneumoniae
. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two
rpsL
alleles in a process that depended on
recA
and that was susceptible to correction by mismatch repair.
Endophytic Colonization and In Planta Nitrogen Fixation by a
Herbaspirillum
sp. Isolated from Wild Rice Species
Authors: Elbeltagy Adel , Nishioka Kiyo , Sato Tadashi , Suzuki Hisa , Ye Bin , Hamada Toru , Isawa Tsuyoshi , Mitsui Hisayuki , Minamisawa Kiwamu
Published: July 2002

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera:
Herbaspirillum
,
Ideonella
,
Enterobacter
, and
Azospirillum
. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the
Herbaspirillum
genus. To examine whether
Herbaspirillum
sp. strain B501 isolated from wild rice,
Oryza officinalis
, endophytically colonizes rice plants, the
gfp
gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice
Oryza sativa
, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that
Herbaspirillum
sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice,
O. officinalis
, was detected by the acetylene reduction and
15
N
2
gas incorporation assays. Therefore, we conclude that
Herbaspirillum
sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly
O. officinalis
.
Development of a Gene Cloning and Inactivation System for Halorespiring
Desulfitobacterium dehalogenans
Authors: Smidt Hauke , van der Oost John , de Vos Willem M.
Published: July 2002

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium
Desulfitobacterium dehalogenans
. An electroporation-based transformation procedure resulting in approximately 10
3
to 10
4
transformants per μg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG
+
host9 was shown to replicate at a permissive temperature of 30°C, whereas the replicon was not functional at 40°C. The
D. dehalogenans frdCAB
operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG
+
host9 carrying a 0.6-kb internal
frdA
fragment. Single-crossover integration at the
frdA
locus occurred at a frequency of 3.3 × 10
−4
per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in
D. dehalogenans
and allow for its further exploitation as a dedicated degrader.
Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on theClostridium botulinumNeurotoxin Type A
Authors: Wu Han-Chung , Yeh Chia-Tsui , Huang Yue-Ling , Tarn Lih-Jeng , Lung Chien-Cheng
Published: July 2002

Clostridium botulinumneurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103and 104times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.
Isolation and In Vivo and In Vitro Antifungal Activity of Phenylacetic Acid and Sodium Phenylacetate from
Streptomyces humidus
Authors: Hwang Byung Kook , Lim Song Won , Kim Beom Seok , Lee Jung Yeop , Moon Surk Sik
Published: July 2002

The antifungal substances SH-1 and SH-2 were isolated from
Streptomyces humidus
strain S5-55 cultures by various purification procedures and identified as phenylacetic acid and sodium phenylacetate, respectively, based on the nuclear magnetic resonance, electron ionization mass spectral, and inductively coupled plasma mass spectral data. SH-1 and SH-2 completely inhibited the growth of
Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, Saccharomyces cerevisiae
, and
Pseudomonas syringae
pv. syringae at concentrations from 10 to 50 μg/ml. The two compounds were as effective as the commercial fungicide metalaxyl in inhibiting spore germination and hyphal growth of
P. capsici
. However, the in vivo control efficacies of the two antifungal compounds against
P. capsici
infection on pepper plants were similar to those of H
3
PO
3
and fosetyl-AI but less than that of metalaxyl.
Photoreactivation in Airborne
Mycobacterium parafortuitum
Authors: Peccia Jordan , Hernandez Mark
Published: July 2002

Photoreactivation was observed in airborne
Mycobacterium parafortuitum
exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne
M. parafortuitum
cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne
M. parafortuitum
and
Serratia marcescens
increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.
Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Bacteria
Authors: Aminov R. I. , Chee-Sanford J. C. , Garrigues N. , Teferedegne B. , Krapac I. J. , White B. A. , Mackie R. I.
Published: July 2002

Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H
+
antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this
tet
subcluster unifies 11 genes encoding
tet
efflux pumps and includes
tet
(A),
tet
(B),
tet
(C),
tet
(D),
tet
(E),
tet
(G),
tet
(H),
tet
(J),
tet
(Y),
tet
(Z), and
tet
(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control
tet
templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded
tet
fingerprint of animal feed was found to be
tetCDEHZ
, while the corresponding fingerprint of total intestinal microbiota was
tetBCGHYZ
. Interestingly, the
tet
fingerprints in geographically distant waste lagoons were identical (
tetBCEHYZ
) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of
tet
genes in comparison with the fecal diversity could be detected, suggesting that the
tet
pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The
tet
efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of
tet
genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.
Development, Characterization, and Diagnostic Applications of Monoclonal Antibodies against Bovine Rotavirus
Authors: Al-Yousif Yousif , Al-Majhdi Fahad , Chard-Bergstrom Cindy , Anderson Joe , Kapil Sanjay
Published: July 2002

Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.
Porins and Lipopolysaccharide Induce Apoptosis in Human Spermatozoa
Authors: Gorga Fernanda , Galdiero Marilena , Buommino Elisabetta , Galdiero Emilia
Published: July 2002

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted fromSalmonella entericaserovar Typhimurium andPasteurella multocidaand were mixed with human spermatozoa for detection of levels of apoptosis.
Biological Response Modifier Activity of an Exopolysaccharide fromPaenibacillus jamilaeCP-7
Authors: Ruiz-Bravo Alfonso , Jimenez-Valera Maria , Moreno Encarnacion , Guerra Victor , Ramos-Cormenzana Alberto
Published: July 2002

An extracellular polysaccharide was purified from culture supernatants ofPaenibacillus jamilaeCP-7, a gram-positive bacillus that was isolated from compost prepared with olive mill wastewaters. The extracellular polysaccharide was produced under aerobic conditions in a medium containing olive mill wastewaters (80% [vol/vol]). This exopolymer had a low level of acute toxicity when it is administered to BALB/c mice by the intraperitoneal route. Interesting immunomodulatory effects were detected when mice were given 10 mg of exopolysaccharide per kg of body weight; the proliferative responses of splenocytes to B-cell and T-cell mitogens were suppressed, the in vitro levels of production of gamma interferon and granulocyte-macrophage colony-stimulating factor by unstimulated and lipopolysaccharide-stimulated splenocytes were enhanced, and the levels of resistance to the intracellular pathogenListeria monocytogeneswas increased in mice. Also, the exopolysaccharide was able to induce lymphocyte proliferation in vitro. We conclude thatP. jamilaeproduces an exopolysaccharide with interesting immunomodulatory properties.
STAT4 Is Required for Antibacterial Defense but Enhances Mortality during Polymicrobial Sepsis
Authors: Godshall Christopher J. , Lentsch Alex B. , Peyton James C. , Scott Melanie J. , Cheadle William G.
Published: July 2002

The signal transducer and activator of transcription factor 4 (STAT4) pathway mediates the intracellular effects of interleukin-12 (IL-12), leading to the production of gamma interferon, induction of a T helper type 1 response, and increased natural killer cell cytotoxicity. The purpose of this study was to determine the role of the STAT4 pathway during polymicrobial peritonitis in the cecal ligation and puncture (CLP) model. CLP was performed on STAT4-deficient (STAT4−/−) and wild-type control (BALB/c) mice. At 4 h after CLP, STAT4−/−mice had significantly higher bacterial counts in the peritoneal lavage fluid, liver, and blood. This difference persisted for 18 h in the peritoneal lavage fluid and blood. Neutrophil migration to the site of infection and into remote tissues was unaffected. Despite higher bacterial counts locally and systemically, STAT4−/−mice had a lower mortality rate than BALB/c controls. In contrast, blockade of IL-12 in BALB/c mice was detrimental to host survival. A blunted serum IL-12 response at 18 h after CLP was exhibited in STAT4−/−mice. These results suggest several critical roles for the STAT4 pathway in the resolution of polymicrobial infections. Additionally, the disparate effects observed with IL-12 blockade and STAT4 deficiency on host survival suggest that IL-12 may activate alternate pathways promoting survival.
Cytokine Profiles of Pediatric Patients Treated with Antibiotics for Pyelonephritis: Potential Therapeutic Impact
Authors: Kassir Kari , Vargas-Shiraishi Ofelia , Zaldivar Frank , Berman Monique , Singh Jasjit , Arrieta Antonio
Published: July 2002

Urinary tract infections are common in infants and children. Pyelonephritis may result in serious complications, such as renal scarring, hypertension, and renal failure. Identification of the timing of release of inflammatory cytokines in relation to pyelonephritis and its treatment is essential for designing interventions that would minimize tissue damage. To this end, we measured urinary cytokine concentrations of interleukin-1β (IL-1β), IL-6, and IL-8 in infants and children with pyelonephritis and in healthy children. Children that presented to our institution with presumed urinary tract infection were given the diagnosis of pyelonephritis if they had a positive urine culture, pyuria, and one or more of the following indicators of systemic involvement: fever, elevated peripheral white blood cell count, or elevated C-reactive protein. Urine samples were obtained at the time of presentation prior to the administration of antibiotics, immediately after completion of the first dose of antibiotics, and at follow up 12 to 24 h after presentation. IL-1β, IL-6, and IL-8 concentrations were measured by enzyme-linked immunosorbent assay. Creatinine concentrations were also determined, and cytokine/creatinine ratios were calculated to standardize samples. Differences between preantibiotic and follow-up cytokine/creatinine ratios were significant for IL-1β, IL-6, and IL-8 (
P
< 0.01). Differences between preantibiotic and control cytokine/creatinine ratios were also significant for IL-1β, IL-6, and IL-8 (
P
< 0.01). Our study revealed that the urinary tract cytokine response to infection is intense but dissipates shortly after the initiation of antibiotic treatment. This suggests that renal damage due to inflammation begins early in infection, underscoring the need for rapid diagnosis and intervention.
Exposure of HEp-2 Cells to Stress Conditions Influences Antinuclear Antibody Reactivity
Authors: Du Liping , Fukushima Sachiko , Sallmyr Annahita , Manthorpe Rolf , Bredberg Anders
Published: July 2002

This study of stress-related antinuclear antibody (ANA) reactivity was undertaken with the objective of improving clinical ANA testing. ANA was determined by parallel enzyme-linked immunosorbent assays of crude nuclear protein antigen extracted from HEp-2 cells either grown under optimal conditions (providing nonstress ANA antigen) or exposed to stress (providing stress ANA antigen). The stress stimuli used were gamma radiation (causing DNA damage) and a hypertonic environment (causing apoptosis). Signs of stress-related ANA reactivity were seen among connective tissue disease (CTD) patients (including patients with systemic lupus erythematosus; mixed CTD; calcinosis, Reynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia; scleroderma; and Sjögren's syndrome): 11% showed stress-positive ANA (i.e., a significantly stronger ANA reactivity with the extract from stressed cells), whereas 21% showed a markedly weaker reaction with the stress antigen. In contrast, among ANA screening patient sera, with no diagnosis of CTD, the fraction showing stress-positive ANA was higher (7 to 8%, depending on the type of stress) than among those showing a lower reactivity with stress antigen (1.5 to 2.5%). Only one serum among 89 (1%) tested sera from healthy individuals showed a stress-related ANA reaction. This demonstration of stress-related ANA suggests a means to improve the performance of clinical ANA testing.
Helicobacter pylori
Seroprevalence in Asymptomatic Pregnant Women in France
Authors: Kalach N. , Desramé J. , Bonnet C. , Commegeille P. , Couturier D. , Chaussade S. , Hance P. , Dupont C. , Raymond J.
Published: July 2002

The purpose of our study was to evaluate the incidence of
Helicobacter pylori
seropositivity in two different populations of asymptomatic pregnant women from different geographic origins during two separate time periods. A retrospective study of consecutive sera obtained from 169 and 302 asymptomatic pregnant women in 1990 and 1999, respectively, was carried out. The global
H. pylori
seroprevalences for 1990 and 1999 were 21.3 and 21.5% (where
P
is nonsignificant), respectively. For both periods the
H. pylori
seroprevalences were significantly higher in non-French pregnant women (66.6 and 50.6%) than in French pregnant women (18.7 and 11.2%) (
P
= 0.01 and 0.001, respectively).
H. pylori
seroprevalence in French pregnant women decreased significantly from the first period (18.7%) to the second one (11.2%) (
P
= 0.03).
CD8+T-Cell Priming against a NonsecretedListeria monocytogenesAntigen Is Independent of the Antimicrobial Activities of Gamma Interferon
Authors: Tvinnereim Amy R. , Harty John T.
Published: July 2002

Sublethal infection of mice with recombinantListeria monocytogenesexpressing a model epitope in either secreted or nonsecreted form results in similar CD8+T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-γ), a cytokine made by multiple cell types in response toL. monocytogenesinfection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-γ knockout (KO) mice were used to address the requirement for IFN-γ in CD8+T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecretedL. monocytogenesantigens. We demonstrate that IFN-γ KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8+T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primaryL. monocytogenesinfection, previously immunized and naive IFN-γ KO mice were able to generate CD8+T-cell responses against both secreted and nonsecretedL. monocytogenesantigens which were similar to responses of WT mice. Interestingly, IFN-γ KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-γ KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-γ are not required for priming of CD8+T cells by cross-presentation of a model exogenous antigen or in response to a nonsecretedL. monocytogenesantigen. In addition, the capacity of IFN-γ to activate the microbicidal activities of macrophages is not required for the very early innate immune response toL. monocytogenesor priming of CD8+T cells against a nonsecreted bacterial antigen.
Lipopolysaccharide-Induced Gelatinase Granule Mobilization Primes Neutrophils for Activation by Galectin-3 and Formylmethionyl-Leu-Phe
Authors: Almkvist Jenny , Fäldt Jenny , Dahlgren Claes , Leffler Hakon , Karlsson Anna
Published: July 2002

We have earlier shown that galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, induces activation of the NADPH oxidase in exudated but not in peripheral blood neutrophils (A. Karlsson, P. Follin, H. Leffler, and C. Dahlgren, Blood 91:3430–3438, 1998). The alteration in responsiveness occurring during extravasation correlated with mobilization of the gelatinase and/or specific granules to the cell surface, indicating a role for mobilizable galectin-3 receptors. In this study we have investigated galectin-3-induced NADPH oxidase activation, measured as superoxide production, in lipopolysaccharide (LPS)-primed neutrophils. Upon galectin-3 challenge, the LPS-primed cells produced superoxide, both extracellularly and intracellularly. A primed extracellular response to formylmethionyl-Leu-Phe (fMLF) was also achieved. The exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the cell surface was markedly increased after LPS treatment, indicating that granule fusion with the plasma membrane had occurred. Further assessment of specific markers for neutrophil granules showed that the LPS treatment had mobilized the gelatinase granules but only a minor fraction of the specific granules. We thus suggest that the mechanism behind LPS priming lies at the level of granule (receptor) mobilization for galectin-3 as well as for fMLF.
Mutagenesis and Functional Reconstitution of Chlamydial Major Outer Membrane Proteins: VS4 Domains Are Not Required for Pore Formation but Modify Channel Function
Authors: Hughes E. S. , Shaw K. M. , Ashley R. H.
Published: July 2002

Chlamidial organisms are obligate intracellular pathogens containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they cluster within four relatively short variable (VS) domains. If MOMPs adopt a β-barrel fold, like bacterial porins, the VS domains may form extramembranous loops and the conserved regions of the protein may correspond to predicted membrane-located β-strands. However, molecular studies on native MOMPs have been hampered by the need to culture chlamydiae in eukaryotic host cells and purification and reconstitution remain problematic. In addition, the organisms are difficult to manipulate genetically, and it has also been difficult to functionally reconstitute recombinant MOMPs. To help overcome these problems and improve our understanding of MOMP structure and function, we cloned and expressedC. trachomatisandC. psittaciMOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we tested the hypothesis that the largest variable domain forms an extramembranous loop that contributes to these differences. Proteins in which VS4 was deleted continued to form functional ion channels, consistent with the idea that the domain forms an extramembranous protein loop and incompatible with models in which it contributes to predicted membrane-located β-strands. Additionally, the properties of the chimeric proteins strongly suggested that the VS4 domain interacts closely with other regions of the protein to form the channel entrance or vestibule. Our approach can be used to probe structure-function relationships in chlamydial MOMPs and may have implications for the generation of effective antichlamydial vaccines.
Selection of Virulence-Associated Determinants of
Streptococcus suis
Serotype 2 by In Vivo Complementation
Authors: Smith Hilde E. , Buijs Herma , Wisselink Henk J. , Stockhofe-Zurwieden Norbert , Smits Mari A.
Published: July 2002

Within
Streptococcus suis
serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. We introduced a genomic library of a pathogenic strain into a weakly pathogenic strain. After infection of the library into young piglets pathogenic transformants were selected. One specific transformant containing a 3-kb fragment of the pathogenic strain appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weakly pathogenic strains, increased the virulence of these strains considerably. In contrast, introduction of the corresponding fragment of a weakly pathogenic strain had only minor effects on virulence. Nucleotide sequence analysis of the selected fragment of the pathogenic strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weakly pathogenic strain. These data strongly suggest that the selected fragment contains determinants important for virulence.
Borrelia burgdorferi
Proteins Whose Expression Is Similarly Affected by Culture Temperature and pH
Authors: Ramamoorthy Ramesh , Scholl-Meeker Dorothy
Published: July 2002

Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, of
Borrelia burgdorferi
in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34°C but not at 24°C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of
B. burgdorferi
proteins, which in culture is selectively expressed under conditions of 34°C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated.
Distinct Regulatory Pathways Control Expression of
Borrelia burgdorferi
Infection-Associated OspC and Erp Surface Proteins
Authors: Babb Kelly , El-Hage Nazira , Miller Jennifer C. , Carroll James A. , Stevenson Brian
Published: July 2002

Deciphering the mechanisms by which
Borrelia burgdorferi
controls the synthesis of proteins associated with mammalian infection will be an important step toward understanding the pathogenic properties of Lyme disease-causing bacteria. We present results of studies indicating that
B. burgdorferi
senses a wide variety of environmental stimuli, including soluble chemicals, which enables it to independently control synthesis of the Erp and OspC proteins. Regulation of OspC and Erp expression appears to occur at the level of transcription. In this regard, we observed that one or more DNA-binding proteins interact specifically with
erp
promoter DNA but not with the
ospC
promoter.
Dendritic Cell Activation and Cytokine Production Induced by Group BNeisseria meningitidis: Interleukin-12 Production Depends on Lipopolysaccharide Expression in Intact Bacteria
Authors: Dixon Garth L. J. , Newton Phillippa J. , Chain Benjamin M. , Katz David , Andersen Svein Rune , Wong Simon , van der Ley Peter , Klein Nigel , Callard Robin E.
Published: July 2002

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain ofNeisseria meningitidiscompared to an isogenic mutantlpxAstrain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain,lpxAmutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent andlpxAstrains induced production of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12. Addition of exogenous LPS to thelpxAstrain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components ofN. meningitidisinduce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components ofN. meningitidisas potential vaccine candidates.
Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein
Authors: Matsunaga James , Young Tracy A. , Barnett Jeanne K. , Barnett Dean , Bolin Carole A. , Haake David A.
Published: July 2002

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulentLeptospira kirschneriserovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that inEscherichia coliexpressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel ofLeptospirastrains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45is a peripheral membrane protein. Finally, we found that P31LipL45levels increased asLeptospiraentered the stationary phase, indicating that P31LipL45levels were regulated. Hamsters infected withL. kirschneriformed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed byL. kirschnerithat colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
Role of the Dermonecrotic Toxin of
Bordetella bronchiseptica
in the Pathogenesis of Respiratory Disease in Swine
Authors: Brockmeier Susan L. , Register Karen B. , Magyar Tibor , Lax Alistair J. , Pullinger Gillian D. , Kunkle Robert A.
Published: July 2002

Bordetella bronchiseptica
is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic
dnt
mutants to their virulent DNT
+
parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the
dnt
mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the
dnt
mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the
dnt
mutant strains. Therefore, production of DNT by
B. bronchiseptica
is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.
Modulation of Immune Responses toMycobacterium bovisin Cattle Depleted of WC1+γδ T Cells
Authors: Kennedy Hilary E. , Welsh Michael D. , Bryson David G. , Cassidy Joseph P. , Forster Fiona I. , Howard Christopher J. , Collins Robert A. , Pollock John M.
Published: July 2002

It is accepted that cell-mediated immune responses predominate in mycobacterial infections. Many studies have shown that CD4+T cells produce Th1 cytokines, such as gamma interferon (IFN-γ), in response to mycobacterial antigens and that the cytolytic activity of CD8+cells toward infected macrophages is important. However, the extent and manner in which γδ T cells participate in this response remain unclear. In ruminants, γδ T cells comprise a major proportion of the peripheral blood mononuclear cell population. We have previously shown that WC1+γδ T cells are involved early inMycobacterium bovisinfection of cattle, but their specific functions are not well understood. Here we describe an in vivo model of bovine tuberculosis in which the WC1+γδ T cells were depleted from the peripheral circulation and respiratory tract, by infusion of WC1+-specific monoclonal antibody, prior to infection. While no effects on disease pathology were observed in this experiment, results indicate that WC1+γδ T cells, which become significantly activated (CD25+) in the circulation of control calves from 21 days postinfection, may play a role in modulating the developing immune response toM. bovis. WC1+-depleted animals exhibited decreased antigen-specific lymphocyte proliferative response, an increased antigen-specific production of interleukin-4, and a lack of specific immunoglobulin G2 antibody. This suggests that WC1+γδ TCR+cells contribute, either directly or indirectly, toward the Th1 bias of the immune response in bovine tuberculosis—a hypothesis supported by the decreased innate production of IFN-γ, which was observed in WC1+-depleted calves.
Stachylysin May Be a Cause of Hemorrhaging in Humans Exposed to
Stachybotrys chartarum
Authors: Vesper Stephen J. , Vesper Mary Jo
Published: July 2002

Stachybotrys chartarum
is a toxigenic fungus that has been associated with human health concerns such as nasal bleeding in adults and pulmonary hemosiderosis (PH) in infants. Seven of eight strains of
S. chartarum
isolated from homes of infants with PH in Cleveland, Ohio, and the strain from the lung of an infant with PH in Texas produced stachylysin in tryptic soy broth (TSB), whereas only one out of eight strains isolated from control homes produced stachylysin. However, all strains produced stachylysin when grown on TSB with 0.7% sheep's blood. When stachylysin was injected into
Lumbricus terrestis
, the erythrocruorin hemoglobin (absorbance peaks at 280 and 415 nm) was released, resulting in a lethal effect. These results support the hypothesis that stachylysin may be one agent responsible for hemorrhaging in humans.
Role of Nitric Oxide in Host Defense in Murine Salmonellosis as a Function of Its Antibacterial and Antiapoptotic Activities
Authors: Alam Mohammad Samiul , Akaike Takaaki , Okamoto Shinichiro , Kubota Tatsuo , Yoshitake Jun , Sawa Tomohiro , Miyamoto Yoichi , Tamura Fumio , Maeda Hiroshi
Published: July 2002

Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulentSalmonella entericaserovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD50s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 × 104CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD50, 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD50, 6 × 102CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute toSalmonellapathogenicity. These results indicate that NO has significant host defense functions inSalmonellainfections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.
Cryptic and Exposed Invariable Regions of VlsE, the Variable Surface Antigen ofBorrelia burgdorferisl
Authors: Liang Fang Ting , Nowling Jena M. , Philipp Mario T.
Published: July 2002

Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies ofB. burgdorferisensu lato. IR6is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR2to IR5) were investigated. Antisera to synthetic peptides based on each of the IR2to IR5sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR2, IR4, and IR5but not to IR3, indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.
The N-Terminal Region of the
Escherichia coli
WecA (Rfe) Protein, Containing Three Predicted Transmembrane Helices, Is Required for Function but Not for Membrane Insertion
Authors: Amer Amal O. , Valvano Miguel A.
Published: July 2002

The correct site for translation initiation for
Escherichia coli
WecA (Rfe), presumably involved in catalyzing the transfer of
N
-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.
Escherichia coli
Responses to a Single DNA Adduct
Authors: Pandya Gagan A. , Yang In-Young , Grollman Arthur P. , Moriya Masaaki
Published: July 2002

To study the mechanisms by which
Escherichia coli
modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various
E. coli
pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1,
N
6
-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional
recA
,
recF
,
recO
, and
recR
genes, suggesting the mechanism to be daughter strand gap repair; (iii) the
ruvABC
genes or the
recG
gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.
Mutagenesis and Functional Characterization of the
glnB
,
glnA
, and
nifA
Genes from the Photosynthetic Bacterium
Rhodospirillum rubrum
Authors: Zhang Yaoping , Pohlmann Edward L. , Ludden Paul W. , Roberts Gary P.
Published: July 2002

Nitrogen fixation is tightly regulated in
Rhodospirillum rubrum
at two different levels: transcriptional regulation of
nif
expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of
glnB
,
glnA
, and
nifA
mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect
glnB
(structural gene for P
II
) were constructed. While P
II
-Y51F showed a lower nitrogenase activity than that of wild type, a P
II
deletion mutant showed very little
nif
expression. This effect of P
II
on
nif
expression is apparently the result of a requirement of P
II
for NifA activation, whose activity is regulated by NH
4
+
in
R. rubrum
. The modification of glutamine synthetase (GS) in these
glnB
mutants appears to be similar to that seen in wild type, suggesting that a paralog of P
II
might exist in
R. rubrum
and regulate the modification of GS. P
II
also appears to be involved in the regulation of DRAT activity, since an altered response to NH
4
+
was found in a mutant expressing P
II
-Y51F. The adenylylation of GS plays no significant role in
nif
expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH
4
+
and darkness treatments.
Kinetics and Substrate Specificity of Membrane-Reconstituted Peptide Transporter DtpT of
Lactococcus lactis
Authors: Fang Gang , Konings Wil N. , Poolman Bert
Published: July 2002

The peptide transport protein DtpT of
Lactococcus lactis
was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[
14
C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, ω-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the
l
configuration, and
trans
-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT
1
and PepT
2
shows that the bacterial transporter is more restrictive in its substrate recognition.