bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability
Authors: Ranjard L. , Poly F. , Lata J.-C. , Mougel C. , Thioulouse J. , Nazaret S.
Published: July 2002

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.
Visualization of
N
-Acylhomoserine Lactone-Mediated Cell-Cell Communication between Bacteria Colonizing the Tomato Rhizosphere
Authors: Steidle Anette , Sigl Katja , Schuhegger Regina , Ihring Alexandra , Schmid Markus , Gantner Stephan , Stoffels Marion , Riedel Kathrin , Givskov Michael , Hartmann Anton , Langebartels Christian , Eberl Leo
Published: July 2002

Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing
N
-acyl-
l
-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of
Pseudomonas
putida
IsoF and
Serratia
liquefaciens
MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negative
P
.
putida
strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.
Analysis of the Type IV Fimbrial-Subunit Gene
fimA
of
Xanthomonas hyacinthi
: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths
Authors: van Doorn J. , Hollinger T. C. , Oudega B.
Published: July 2002

A sensitive and specific detection method was developed for
Xanthomonas hyacinthi
; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene
fimA
from strain S148. The
fimA
gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of
fimA
was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as
Xanthomonas campestris
pv. vesicatoria,
Neisseria gonorrhoeae
, and
Moraxella bovis
. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of
fimA
, amplified a 226-bp DNA fragment in all
X. hyacinthi
isolates. This PCR was shown to be pathovar specific, as assessed by testing 71
Xanthomonas
pathovars and bacterial isolates belonging to other genera, such as
Erwinia
and
Pseudomonas
. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in
X. hyacinthi
. Only two
Xanthomonas translucens
pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the
fimA
gene of
X. campestris
pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280–1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of
X. hyacinthi
per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for
X. hyacinthi
with PCR.
Characterization of
Listeria monocytogenes
Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fingerprinting Techniques
Authors: Franciosa Giovanna , Tartaro Stefania , Wedell-Neergaard Christina , Aureli Paolo
Published: July 2002

A total of 32
Listeria monocytogenes
strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated
L. monocytogenes
food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.
Natural Transformation of
Pseudomonas fluorescens
and
Agrobacterium tumefaciens
in Soil
Authors: Demanèche Sandrine , Kay Elisabeth , Gourbière François , Simonet Pascal
Published: July 2002

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria,
Agrobacterium tumefaciens
and
Pseudomonas fluorescens
, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover,
P. fluorescens
produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested.
A. tumefaciens
could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.
The Leucine Incorporation Method Estimates Bacterial Growth Equally Well in Both Oxic and Anoxic Lake Waters
Authors: Bastviken David , Tranvik Lars
Published: July 2002

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O
2
contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O
2
contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O
2
contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O
2
-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.
Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in
Acinetobacter
sp. Strain BD413 and
Thermus thermophilus
HB27
Authors: Friedrich Alexandra , Hartsch Thomas , Averhoff Beate
Published: July 2002

The mesophile
Acinetobacter
sp. strain BD413 and the extreme thermophile
Thermus thermophilus
HB27 display high frequencies of natural transformation. In this study we identified and characterized a novel competence gene in
Acinetobacter
sp. strain BD413,
comA
, whose product displays significant similarities to the competence proteins ComA and ComEC in
Neisseria
and
Bacillus
species. Transcription of
comA
correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery. This finding strongly suggests that
comA
is part of a competence regulon. Examination of the genome sequence of
T. thermophilus
HB27 led to detection of a
comA/comEC
-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the
Bacillus subtilis
competence protein ComEA. To examine whether these two ORFs, designated
comEC
and
comEA
, are implicated in natural transformation of
T. thermophilus
HB27, both were disrupted by using a thermostable kanamycin resistance marker. Natural transformation in
comEC
mutants was reduced 1,000-fold, whereas in
comEA
mutants the natural transformation phenotype was completely eliminated. These results strongly suggest that both genes,
comEC
and
comEA
, are required for natural transformation in
T. thermophilus
HB27. Several transmembrane α-helices are predicted based on the amino acid sequences of ComA in
Acinetobacter
sp. strain BD413 and ComEC in
T. thermophilus
HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane.
Effects of Iron Limitation on the Degradation of Toluene by
Pseudomonas
Strains Carrying the TOL (pWWO) Plasmid
Authors: Dinkla Inez J. T. , Gabor Esther M. , Janssen Dick B.
Published: July 2002

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by
Pseudomonas putida
mt2 and the transconjugant rhizosphere bacterium
P. putida
WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three
meta
-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer
o
-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.
Efficient System for Directed Integration into the
Lactobacillus acidophilus
and
Lactobacillus gasseri
Chromosomes via Homologous Recombination
Authors: Russell W. M. , Klaenhammer T. R.
Published: July 2002

An efficient method is described for the generation of site-specific chromosomal integrations in
Lactobacillus acidophilus
and
Lactobacillus gasseri
. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35–50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the
Lactobacillus acidophilus
NCFM
lacL
gene encoding β-galactosidase and of the
Lactobacillus gasseri
ADH
gusA
gene encoding β-glucuronidase.
Molecular Characterization of a Thermostable Cyanophycin Synthetase from the Thermophilic Cyanobacterium
Synechococcus
sp. Strain MA19 and In Vitro Synthesis of Cyanophycin and Related Polyamides
Authors: Hai Tran , Oppermann-Sanio Fred Bernd , Steinbüchel Alexander
Published: July 2002

The thermophilic cyanobacterium
Synechococcus
sp. strain MA19 contained the structural genes for cyanophycin synthetase (
cphA
) and cyanophycinase (
cphB
), which were identified, cloned, and sequenced in this study. The translation products of
cphA
and
cphB
exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as
Anabaena variabilis
and
Synechocystis
sp. Recombinant cells of
Escherichia coli
harboring
cphA
colinear with
lacPO
accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-β-
d
-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates,
l
-arginine and
l
-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, α-arginyl aspartic acid dipeptide, and poly-α,β-
dl
-aspartic acid as primers and also incorporated β-hydroxyaspartic acid instead of
l
-aspartic acid or
l
-canavanine instead of
l
-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.
Bacteriocin-Like Activity of
Butyrivibrio fibrisolvens
JL5 and Its Effect on Other Ruminal Bacteria and Ammonia Production
Authors: Rychlik Jennifer L. , Russell James B.
Published: July 2002

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10
−6
. The 10
−8
and 10
−9
dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited
Clostridium sticklandii
SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to
Butyrivibrio fibrisolvens
B835. Strain JL5 inhibited
B. fibrisolvens
49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of
B. fibrisolvens
49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of
C. sticklandii
SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and
C. sticklandii
SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.
Widespread Distribution in Polar Oceans of a 16S rRNA Gene Sequence with Affinity to
Nitrosospira
-Like Ammonia-Oxidizing Bacteria
Authors: Hollibaugh James T. , Bano Nasreen , Ducklow Hugh W.
Published: July 2002

We analyzed the phylogenetic compositions of ammonia-oxidizing bacteria of the β subclass of
Proteobacteria
from 42 Southern Ocean samples. We found a
Nitrosospira
-like 16S rRNA gene sequence in all 20 samples that yielded PCR products (8 of 30 samples from the Ross Sea and 12 of 12 samples from the Palmer Peninsula). We also found this sequence in Arctic Ocean samples, indicating a transpolar, if not global, distribution; however, slight differences between Arctic and Antarctic sequences may be evidence of polar endemism.
Biodegradation of
cis
-Dichloroethene as the Sole Carbon Source by a β-Proteobacterium
Authors: Coleman Nicholas V. , Mattes Timothy E. , Gossett James M. , Spain Jim C.
Published: July 2002

An aerobic bacterium capable of growth on
cis-
dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from
Polaromonas vacuolata
, indicating that the isolate was a β-proteobacterium. At 20°C, strain JS666 grew on cDCE with a minimum doubling time of 73 ± 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 ± 0.2 μM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride,
trans
-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.
Use of an Attenuated Leishmanial Parasite as an Immunoprophylactic and Immunotherapeutic Agent against Murine Visceral Leishmaniasis
Authors: Mukhopadhyay Srirupa , Bhattacharyya Sandip , Majhi Ramdhan , De Tripti , Naskar Khudiram , Majumdar Subrata , Roy Syamal
Published: July 2002

The ability of the leishmanial parasite UR6 to act as an immunoprophylactic and immunotherapeutic agent againstLeishmania donovaniinfection in BALB/c mice was investigated. Unlike the virulentL. donovaniAG83 (MOHOM/IN/1983/AG83), UR6 given through intracardiac route failed to induce visceral infection, but when it was injected subcutaneously, UR6 induced a short-lived and localized self-healing skin lesion. Priming of peritoneal macrophages with UR6 in vitro induced superoxide (O2−) generation, whereas similar experiments with virulent AG83 inhibited O2−generation. It was observed that priming of mice with either live or sonicated UR6 in the absence of any adjuvant provided strong protection against subsequent virulent challenge. Further, UR6-primed infected mice not only displayed a strong antileishmanial delayed-type hypersensitivity (DTH) response but also showed an elevated level of the serum antileishmanial immunoglobulin G2a (IgG2a) isotype, whereas infected mice failed to mount any antileishmanial DTH response and showed an elevated level of IgG1. This indicates that UR6 priming and subsequentL. donovaniinfection allowed the expansion of Th1 cells. Our studies indicate that UR6 has potential to be used as an immunoprophylactic and immunotherapeutic agent against experimental visceral leishmaniasis.
Measurement of Cytokine Secretion, Intracellular Protein Expression, and mRNA in Resting and Stimulated Peripheral Blood Mononuclear Cells
Authors: Sullivan Kathleen E. , Cutilli Joie , Piliero Lisa M. , Ghavimi-Alagha Darlene , Starr Stuart E. , Campbell Donald E. , Douglas Steven D.
Published: July 2002

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-α), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-α, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.
Clarithromycin Attenuates Mastectomy-Induced Acute Inflammatory Response
Authors: Chow Louis W. C. , Yuen Kwok-Yung , Woo Patrick C. Y. , Wei William I.
Published: July 2002

Based on the observation that administration of clarithromycin led to an attenuation of the inflammatory response induced by surgical trauma in a guinea pig model, we investigated the potential beneficial effects of clarithromycin on the local and systemic inflammatory response in patients undergoing mastectomy in an open-label prospective study. During a 16-month period, 54 patients who underwent mastectomy were randomly divided into two groups. In one group, the patients received oral clarithromycin at a dose of 500 mg twice a day, from the day before to 3 days after mastectomy. There was no significant difference in the incidence of antibiotic prophylaxis-related toxicities or postoperative infections between the patients who received clarithromycin and those who did not. Clarithromycin treatment was significantly associated with an attenuation of febrile response, tachycardia, tachypnea, and an increase in monocyte counts (P, <0.0001, <0.01, <0.05, and <0.01, respectively). Clarithromycin also reduced the intensity and duration of postoperative pain (P, <0.05 and <0.005, respectively) and increased the range of motion of the involved shoulder (P< 0.05 for abduction and flexion). We conclude that clarithromycin effectively modulates the acute inflammatory response associated with mastectomy and produces a better clinical outcome.
Lipoteichoic Acid Inhibits Interleukin-2 (IL-2) Function by Direct Binding to IL-2
Authors: Plitnick Lisa M. , Jordan Robert A. , Banas Jeffrey A. , Jelley-Gibbs Dawn M. , Walsh Mary C. , Preissler Mark T. , Gosselin Edmund J.
Published: July 2002

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.
Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B,
Yersinia pestis
-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus
Authors: Smith Darci R. , Rossi Cynthia A. , Kijek Todd M. , Henchal Erik A. , Ludwig George V.
Published: July 2002

The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B,
Yersinia pestis
-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log
10
concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories.
Chlamydia pneumoniae
Exacerbates Aortic Inflammatory Foci Caused by Murine Cytomegalovirus Infection in Normocholesterolemic Mice
Authors: Burian Katalin , Berencsi Klara , Endresz Valeria , Gyulai Zsofia , Valyi-Nagy Tibor , Valyi-Nagy Istvan , Bakay Marta , Geng Yuemei , Virok Dezso , Kari Laszlo , Hajnal-Papp Rozalia , Trinchieri Giorgio , Gonczol Eva
Published: July 2002

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with
Chlamydia pneumoniae
.
Potential Benefit of Plasma Exchange in Treatment of Severe Icteric Leptospirosis Complicated by Acute Renal Failure
Authors: Tse Kai-Chung , Yip Pok-Siu , Hui King-Men , Li Fu-Keung , Yuen Kwok-Yung , Lai Kar-Neng , Chan Tak-Mao
Published: July 2002

Leptospirosis is a common zoonosis seen worldwide, but it is rare in our locality (Hong Kong). Clinical manifestations of leptospirosis are variable and may range from subclinical infection to fever, jaundice, hemorrhagic tendency, and fulminant hepato-renal failure. Severe hyperbilirubinemia and acute renal failure have been associated with high mortality. We report our experience with a patient who developed severe Weil's syndrome with marked conjugated hyperbilirubinemia and oliguric acute renal failure. These complications persisted despite treatment with penicillin and hemodiafiltration. Plasma exchange was instituted in view of the severe hyperbilirubinemia (970 μmol/liter). This was followed by prompt clinical improvement, with recovery of liver and renal function. The beneficial effects of plasma exchange could be attributed to amelioration of the toxic effects of hyperbilirubinemia on hepatocyte and renal tubular cell function. We conclude that plasma exchange should be considered as an adjunctive therapy for patients with severe icteric leptospirosis complicated by acute renal failure who have not shown rapid clinical response to conventional treatment.
Role of Novel Choline Binding Proteins in Virulence of
Streptococcus pneumoniae
Authors: Gosink Khoosheh K. , Mann Elizabeth R. , Guglielmo Chris , Tuomanen Elaine I. , Masure H. Robert
Published: July 2002

The choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall of
Streptococcus pneumoniae
by a conserved choline binding domain. Six new members of this family were identified, and these six plus two recently described cell wall hydrolases, LytB and LytC, were characterized for their roles in virulence. CBP-deficient mutants were constructed and tested for adherence to eukaryotic cells, colonization of the rat nasopharynx, and ability to cause sepsis. Five CBP mutants, CbpD, CbpE, CbpG, LytB, and LytC, showed significantly reduced colonization of the nasopharynx. For CbpE and -G this was attributable to a decreased ability to adhere to human cells. CbpG, a putative serine protease, also played a role in sepsis, the first observation of a pneumococcal virulence determinant strongly operative both on the mucosal surface and in the bloodstream.
Regulation of the
Porphyromonas gingivalis fimA
(Fimbrillin) Gene
Authors: Xie Hua , Chung Whasun O. , Park Yoonsuk , Lamont Richard J.
Published: July 2002

In common with many bacterial virulence genes, the fimbrillin (
fimA
) gene of
Porphyromonas gingivalis
is modulated in response to environmental fluctuation. The
trans
-acting components that comprise the regulatory system for transcriptional activity of the
fimA
gene in
P. gingivalis
were investigated. Three major proteins were found to bind to the upstream region of the
fimA
promoter. One of these proteins was fimbrillin itself, and the other two were a major arginine protease (Rgp) and lysine protease (Kgp). Production of these proteins was necessary for maximal
fimA
transcription. An exogenous
fimA
promoter-
lacZ
reporter was inactive when introduced into a strain of
P. gingivalis
carrying a mutation in the indigenous
fimA
gene. Furthermore,
fimA
mRNA levels were significantly decreased in
rgp
and
kgp
mutant strains. These data indicate that
P. gingivalis
has evolved multiple levels of control of fimbrial gene expression to enhance its survival in hostile environments.
Identification of Discrete Domains within Gonococcal Transferrin-Binding Protein A That Are Necessary for Ligand Binding and Iron Uptake Functions
Authors: Boulton Ian C. , Yost Mary Kate , Anderson James E. , Cornelissen Cynthia Nau
Published: July 2002

The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenicNeisseriaspp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a β-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the ΔL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.
Enzymatic Properties of Dipeptidyl Aminopeptidase IV Produced by the Periodontal PathogenPorphyromonas gingivalisand Its Participation in Virulence
Authors: Kumagai Yumi , Konishi Kiyoshi , Gomi Tomoharu , Yagishita Hisao , Yajima Ayako , Yoshikawa Masanosuke
Published: July 2002

Porphyromonas gingivalisis a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) fromP. gingivalisW83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. AnEscherichia colistrain overproducingP. gingivalisDPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated fromP. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved inP. gingivalisDPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues ofP. gingivalisDPPIV are involved in the catalytic reaction. DPPIV-deficient mutants ofP. gingivaliswere constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor ofP. gingivalisand may play important roles in the pathogenesis of adult periodontitis induced by the organism.
Construction and Characterization of an
agr
Deletion Mutant of
Staphylococcus epidermidis
Authors: Vuong Cuong , Götz Friedrich , Otto Michael
Published: July 2002

The physiological significance of the accessory gene regulator (
agr
) system of
Staphylococcus epidermidis
was investigated by construction of an
agr
deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The
agr
system of
S. epidermidis
thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for
Staphylococcus aureus
. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the
agr
mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the
agr
mutant.
In Vitro and In Vivo Analyses of Constitutive and In Vivo-Induced Promoters in Attenuated Vaccine and Vector Strains of
Vibrio cholerae
Authors: John Manohar , Crean Thomas I. , Calderwood Stephen B. , Ryan Edward T.
Published: July 2002

The optimal promoter for in vivo expression of heterologous antigens by live, attenuated vaccine vector strains of
Vibrio cholerae
is unclear; in vitro analyses of promoter activity may not accurately predict expression of antigens in vivo. We therefore introduced plasmids expressing the B subunit of cholera toxin (CtxB) under the control of a number of promoters into
V. cholerae
vaccine strain Peru2. We evaluated the
tac
promoter, which is constitutively expressed in
V. cholerae
, as well as the in vivo-induced
V. cholerae
heat shock
htpG
promoter and the in vivo-induced
V. cholerae
iron-regulated
irgA
promoter. The functionality of all promoters was confirmed in vitro. In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of the
tac
promoter (2 to 5 μg/ml/unit of optical density at 600 nm [OD
600
]) and, under low-iron conditions, in strains containing the
irgA
promoter (5 μg/ml/OD
600
). We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB. The vaccine strain expressing CtxB under the control of the
tac
promoter elicited the most prominent specific anti-CtxB responses in vivo (serum immunoglobulin G [IgG],
P
≤ 0.05; serum IgA,
P
≤ 0.05; stool IgA,
P
≤ 0.05; bile IgA,
P
≤ 0.05), despite the finding that the
tac
and
irgA
promoters expressed equivalent amounts of CtxB in vitro. Vibriocidal antibody titers were equivalent in all groups of animals. Our results indicate that in vitro assessment of antigen expression by vaccine and vector strains of
V. cholerae
may correlate poorly with immune responses in vivo and that of the promoters examined, the
tac
promoter may be best suited for expression from plasmids of at least certain heterologous antigens in such strains.
Tumor Necrosis Factor Alpha and Interleukin 1β Up-Regulate Gastric Mucosal Fas Antigen Expression inHelicobacter pyloriInfection
Authors: Houghton JeanMarie , Macera-Bloch Lisa S. , Harrison Lawrence , Kim Kyung H. , Korah Reju M.
Published: July 2002

Fas-mediated gastric mucosal apoptosis is gaining attention as a cause of tissue damage due toHelicobacter pyloriinfection. We explored the effects ofH. pyloridirectly, and the effects of the inflammatory environment established subsequent toH. pyloriinfection, on Fas-mediated apoptosis in a nontransformed gastric mucosal cell line (RGM-1). Exposure toH. pylori-activated peripheral blood mononuclear cells (PBMCs), but notH. pyloriitself, induced Fas antigen (Fas Ag) expression, indicating a Fas-regulatory role for inflammatory cytokines in this system. Of various inflammatory cytokines tested, only interleukin 1β and tumor necrosis factor alpha induced Fas Ag expression, and removal of either of these from the conditioned medium abrogated the response. When exposed to Fas ligand, RGM-1 cells treated with PBMC-conditioned medium underwent massive and rapid cell death, interestingly, with a minimal effect on total cell numbers early on. Cell cycle analysis revealed a substantial increase in S phase cells among cells exposed to Fas ligand, suggesting an increase in their proliferative response. Taken together, these data indicate that the immune environment secondary toH. pyloriinfection plays a critical role in priming gastric mucosal cells to undergo apoptosis or to proliferate based upon their Fas Ag status.
Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released byEscherichia coliBacteria into Serum
Authors: Hellman Judith , Loiselle Paul M. , Tehan Megan M. , Allaire Jennifer E. , Boyle Lenora A. , Kurnick James T. , Andrews David M. , Sik Kim Kwang , Warren H. Shaw
Published: July 2002

Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised toEscherichia coliJ5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.
Increased Levels of Soluble Fas Ligand in Serum in
Plasmodium falciparum
Malaria
Authors: Kern Peter , Dietrich Manfred , Hemmer Christoph , Wellinghausen Nele
Published: July 2002

Levels of soluble Fas ligand (sFasL) in serum were elevated in patients with
Plasmodium falciparum
malaria and showed a significant decline during disease course. sFasL levels that were elevated before antimalarial treatment began correlated significantly with depressed total lymphocyte and T-cell counts. These data suggest that Fas-induced apoptosis might play a role in malaria-associated lymphopenia.
Antibody Interactions with the Capsule ofCryptococcus neoformans
Authors: Feldmesser Marta , Rivera Johanna , Kress Yvonne , Kozel Thomas R. , Casadevall Arturo
Published: July 2002

Monoclonal antibodies to the encapsulated fungusCryptococcus neoformansproduce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). FixingC. neoformanscells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to theC. neoformanscapsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to theC. neoformanspolysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.
Vaccination with Calpain Induces a Th1-Biased Protective Immune Response againstSchistosoma japonicum
Authors: Zhang Renli , Yoshida Ayako , Kumagai Takashi , Kawaguchi Hitoshi , Maruyama Haruhiko , Suzuki Takashi , Itoh Makoto , El-Malky Mohamed , Ohta Nobuo
Published: July 2002

A large subunit of calpain, a calcium-activated neutral proteinase, fromSchistosoma japonicumwas cloned and expressed inEscherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed(P < 0.01). Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-γ) by activated CD4+T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-γ seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promotingS. japonicuminfection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point toS. japonicumcalpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.
Differential Interleukin-8 Response of Intestinal Epithelial Cell Line to Reactogenic and Nonreactogenic Candidate Vaccine Strains of
Vibrio cholerae
Authors: Rodrı́guez Boris L. , Rojas Armando , Campos Javier , Ledon Talena , Valle Edgar , Toledo William , Fando Rafael
Published: July 2002

In this study, we analyzed whether attachment of
Vibrio cholerae
vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of
V. cholerae
resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.