bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Cloning of
gusA
, Encoding a New β-Glucuronidase from
Lactobacillus gasseri
ADH
Authors: Russell W. M. , Klaenhammer T. R.
Published: July 2002

The
gusA
gene, encoding a new β-glucuronidase enzyme, has been cloned from
Lactobacillus gasseri
ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than
Escherichia coli
. A plasmid library of
L. gasseri
chromosomal DNA was screened for complementation of an
E. coli gus
mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of
E. coli
K-12 (EC
3.2.1.31
). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative
L. gasseri
strain by expressing the
gusA
gene, subcloned onto a low-copy-number shuttle vector, from the strong
Lactobacillus
P6 promoter. GusA was also expressed in
E. coli
from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and
gusA
homologs in other
L. gasseri
isolates but not in other
Lactobacillus
species tested.
Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single
Cryptosporidium parvum
Oocysts
Authors: Sturbaum Gregory D. , Reed Carrie , Hoover Paul J. , Jost B. Helen , Marshall Marilyn M. , Sterling Charles R.
Published: July 2002

Concurrent with recent advances seen with
Cryptosporidium parvum
detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of
C. parvum
oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from
C. parvum
,
Cryptosporidium baileyi
, and
Cryptosporidium serpentis
but no other
Cryptosporidium
sp. or protozoan tested. Restriction enzyme digestion with
Vsp
I distinguished between
C. parvum
genotypes 1 and 2. Restriction enzyme digestion with
Dra
II distinguished
C. parvum
from
C. baileyi
and
C. serpentis
. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated
C. parvum
oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.
Protection of Tomato Seedlings against Infection by
Pseudomonas syringae
pv. Tomato by Using the Plant Growth-Promoting Bacterium
Azospirillum brasilense
Authors: Bashan Yoav , de-Bashan Luz E.
Published: July 2002

Pseudomonas syringae
pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium
Azospirillum brasilense
were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the
A. brasilense
population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of
P. syringae
pv. tomato (10
7
versus 10
5
CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of
P. syringae
pv. tomato was 1 order of magnitude greater than that of
A. brasilense
(10
7
versus 10
6
CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the
A. brasilense
population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the
P. syringae
pv. tomato population and significantly decreased disease severity. Challenge with
P. syringae
pv. tomato after
A. brasilense
was established in the leaves further reduced both the population of
P. syringae
pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10
5
to 10
6
CFU/g [dry weight] of root). However,
P. syringae
pv. tomato did not survive in the rhizosphere in the presence of
A. brasilense
. Foliar inoculation of
A. brasilense
after
P. syringae
pv. tomato was established on the leaves did not alleviate bacterial speck disease, and
A. brasilense
did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of
A. brasilense
(>10
8
CFU/g [dry weight] of leaf), decreased the population of
P. syringae
pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that
A. brasilense
be used in prevention programs to combat the foliar bacterial speck disease caused by
P. syringae
pv. tomato.
Chi18A, the Endochitinase in the Cellulosome of the Thermophilic, Cellulolytic Bacterium
Clostridium thermocellum
Authors: Zverlov Vladimir V. , Fuchs Klaus-Peter , Schwarz Wolfgang H.
Published: July 2002

The chitinase gene
chiA
was identified on the
Clostridium thermocellum
genome downstream of the endoglucanase gene
celA
. It contains a catalytic module of glycosyl hydrolase family 18 and a cellulosomal dockerin module. Chi18A hydrolyzes aryl-acetyl-chito-oligosaccharides preferentially. In denaturing electrophoresis of purified cellulosomes, a single chitinase activity band was identified in zymograms and Western blots, indicating that Chi18A is the only chitinase in the cellulosome.
Cytokine Gene Expression Occurs More Rapidly in Stimulated Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Persons
Authors: Breen Elizabeth Crabb , McDonald Matthew , Fan Jiang , Boscardin John , Fahey John L.
Published: July 2002

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV−) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV− and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P< 0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-α]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV− PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-α mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P< 0.05) and TNF-α (P< 0.005), compared to HIV− PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-α) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.
Evaluation of Six Immunoassays for Detection of Dengue Virus-Specific Immunoglobulin M and G Antibodies
Authors: Groen Jan , Koraka Penelopie , Velzing Jans , Copra Cedrick , Osterhaus Albert D. M. E.
Published: July 2002

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.
Simple and Rapid Method for Production of Whole-Virus Antigen for Serodiagnosis of Caprine Arthritis-Encephalitis Virus by Enzyme-Linked Immunosorbent Assay
Authors: Simard Carole , Kibenge Molly Twinomwe , Singh Patricia , Dixon Phyllis
Published: July 2002

Polyethylene glycol (PEG) was used to produce whole-virus antigen derived from tissue culture cells infected with a Canadian strain of caprine arthritis-encephalitis virus. PEG antigen batches were obtained after precipitation and concentration of infected tissue culture material with PEG 8000 and final treatment with sodium dodecyl sulfate. The optimum time of harvest of tissue culture extracted material to produce the maximum amount of viral proteins was determined in roller bottles, after cocultivation of infected and noninfected fetal lamb corneal cells. Samples from day 9 to day 25 postculture were collected and processed. By Western blotting, the optimum time of harvest was found to be day 25 following the coculture. Two large batches of PEG antigen were prepared at the optimum time of harvest. Both batches gave similar results when tested by Western blotting and enzyme-linked immunosorbent assay (ELISA), using reference control sera from infected and noninfected goats. For further testing in ELISA, cutoff values and ratios were determined for PEG batch 1, using 200 known serum samples from goats free of the disease. The PEG antigen batch was compared with an in-house ELISA antigen in a kinetic mode, using 498 serum samples from field goats. The in-house ELISA antigen was produced following two rounds of ultracentrifugation and treatment with sodium dodecyl sulfate (R. A. Heckert, W. B. McNab, S. M. Richardson, and M. R. Briscoe, Can. J. Vet. Res. 56:237–241, 1992). The PEG antigen batch was found suitable for ELISA, with a relative specificity of 100% and a relative sensitivity of 99.4% compared to the in-house ELISA antigen. This method of antigen production for ELISA was found to be rapid, inexpensive, and reliable for the diagnosis of caprine-arthritis encephalitis, without requiring the use of sophisticated laboratory equipment.
Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis
Authors: Priest Jeffrey W. , Li Anna , Khan Mohamad , Arrowood Michael J. , Lammie Patrick J. , Ong Corinne S. , Roberts Jacquelin M. , Isaac-Renton Judith
Published: July 2002

Cryptosporidium parvum
is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of
Cryptosporidium
infection in the population.
Detection of Antibodies to
Babesia equi
in Horses by a Latex Agglutination Test Using Recombinant EMA-1
Authors: Xuan Xuenan , Igarashi Ikuo , Tanaka Tetsuya , Fukumoto Shinya , Nagasawa Hideyuki , Fujisaki Kozo , Mikami Takeshi
Published: July 2002

A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to
Babesia equi
was developed. The LAT was able to differentiate very clearly between sera from
B. equi
-infected horses and sera from
Babesia caballi
-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of
B. equi
infection.
Serum Immunoglobulin G Antibody Responses to
Bordetella pertussis
Lipooligosaccharide and
B.
parapertussis
Lipopolysaccharide in Children with Pertussis and Parapertussis
Authors: Trollfors Birger , Lagergård Teresa , Taranger John , Bergfors Elisabet , Schneerson Rachel , Robbins John B.
Published: July 2002

Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of
Bordetella pertussis
and the lipopolysaccharide (LPS) of
Bordetella parapertussis
were measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.
Detection of Y Chromosome DNA as Evidence of Semen in Cervicovaginal Secretions of Sexually Active Women
Authors: Chomont Nicolas , Grésenguet Gérard , Lévy Michel , Hocini Hakim , Becquart Pierre , Matta Mathieu , Tranchot-Diallo Juliette , Andreoletti Laurent , Carreno Marie-Paule , Kazatchkine Michel D. , Bélec Laurent
Published: July 2002

The detection of traces of semen in cervicovaginal secretions (CVS) from sexually active women practicing unprotected sex is a prerequisite for the accurate study of cervicovaginal immunity. Two semen markers, the prostatic-specific antigen (PSA) and the Y chromosome, were detected in parallel in CVS obtained by a standardized vaginal washing of consecutive women attending the principal medical center for sexually transmitted diseases of Bangui, Central African Republic. PSA was detected by immunoenzymatic capture assay in the cell-free fraction of CVS, and the Y chromosome was detected by a single PCR assay of DNA extracted by silica from the cell fraction (Y PCR). Fifty (19%) cell-free fractions of the 264 β-globin-positive CVS samples were positive for PSA, and 100 (38%) cell fractions of the CVS samples were positive for the Y chromosome. All the 50 (19%) PSA-containing CVS samples were also positive for the Y chromosome. Fifty (19%) CVS samples were positive only for the Y chromosome, with no detectable PSA. The remaining 164 (62%) CVS samples were both PSA and Y chromosome negative. These findings demonstrate that CVS from sexually active women may contain cell-associated semen residues unrecognized by conventional immunoenzymatic assays used to detect semen components. The detection of cell-associated male DNA with a highly sensitive and specific procedure such as Y PCR constitutes a method of choice to detect semen traces in female genital secretions.
Diagnosis ofHelicobacter pyloriInfection in Children: Comparison of a Salivary Immunoglobulin G Antibody Test with the [13C]Urea Breath Test
Authors: Bode G. , Marchildon P. , Peacock J. , Brenner H. , Rothenbacher D.
Published: July 2002

The prevalence ofHelicobacter pyloriinfection in a population-based sample of 477 children (mean age ± standard deviation, 5.8 ± 0.5 years) determined by the [13C]urea breath test ([13C]UBT) was 10.7% (95% confidence interval [CI], 8.1 to 13.8%), and that determined by salivary enzyme-linked immunosorbent assay (ELISA) was 11.9% (95% CI, 9.2 to 15.2%). Compared to the [13C]UBT, the sensitivity and specificity of the salivary ELISA were 80.9% (95% CI, 66.3 to 90.4%) and 95.3% (95% CI, 92.7 to 97.1%), respectively.
One-Step PCR To Distinguish B Virus from Related Primate Alphaherpesviruses
Authors: Hirano Makoto , Nakamura Shin , Mitsunaga Fusako , Okada Maki , Shirahama Shuya , Eberle Richard
Published: July 2002

By adding betaine to the PCR mixture, we previously established a PCR method to amplify a DNA segment of the glycoprotein G gene of B virus (BV) derived from a rhesus macaque. We have found that DNA of other BV strains derived from cynomolgus, pigtail, and lion-tailed macaques can also serve as the template in our PCR assay. Under the same conditions no product was obtained with DNA of simian agent 8 of green monkeys andHerpesvirus papio2 of baboons, or the human herpes simplex viruses types 1 and 2. Thus, this PCR method is useful to discriminate BV from other closely related primate alphaherpesviruses.
Whipple's Disease and “Tropheryma whippelii”
Authors: Dutly Fabrizio , Altwegg Martin
Published: July 2002

Whipple's disease is a rare bacterial infection that may involve any organ system in the body. It occurs primarily in Caucasian males older than 40 years. The gastrointestinal tract is the most frequently involved organ, with manifestations such as abdominal pain, malabsorption syndrome with diarrhea, and weight loss. Other signs include low-grade fever, lymphadenopathy, skin hyperpigmentation, endocarditis, pleuritis, seronegative arthritis, uveitis, spondylodiscitis, and neurological manifestations, and these signs may occur in the absence of gastrointestinal manifestations. Due to the wide variability of manifestations, clinical diagnosis is very difficult and is often made only years or even decades after the initial symptoms have appeared. Trimethoprim-sulfamethoxazole for at least 1 year is usually considered adequate to eradicate the infection. The microbiological diagnosis of this insidious disease is rendered difficult by the virtual lack of culture and serodiagnostic methods. It is usually based on the demonstration of periodic acid-Schiff-positive particles in infected tissues and/or the presence of bacteria with an unusual trilaminar cell wall ultrastructure by electron microscopy. Recently, the Whipple bacteria have been characterized at the molecular level by amplification of their 16S rRNA gene(s). Phylogenetic analysis of these sequences revealed a new bacterial species related to the actinomycete branch which was named “Tropheryma whippelli.” Based on its unique 16S ribosomal DNA (rDNA) sequence, species-specific primers were selected for the detection of the organism in clinical specimens by PCR. This technique is currently used as one of the standard methods for establishing the diagnosis of Whipple's disease. Specific and broad-spectrum PCR amplifications mainly but not exclusively from extraintestinal specimens have significantly improved diagnosis, being more sensitive than histopathologic analysis. However, “T. whippelii” DNA has also been found in persons without clinical and histological evidence of Whipple's disease. It is unclear whether these patients are true asymptomatic carriers or whether differences in virulence exist among strains of “T. whippelii” that might account for the variable clinical manifestations. So far, six different “T. whippelii” subtypes have been found by analysis of their 16S-23S rDNA spacer region. Further studies of the pathogen “T. whippelii” as well as the host immune response are needed to fully understand this fascinating disease. The recent cultivation of the organisms is a promising major step in this direction.
Dracunculiasis (Guinea Worm Disease) and the Eradication Initiative
Authors: Cairncross Sandy , Muller Ralph , Zagaria Nevio
Published: July 2002

Dracunculiasis, also known as guinea worm disease, is caused by the large female of the nematode Dracunculus medinensis, which emerges painfully and slowly from the skin, usually on the lower limbs. The disease can infect animals, and sustainable animal cycles occur in North America and Central Asia but do not act as reservoirs of human infection. The disease is endemic across the Sahel belt of Africa from Mauritania to Ethiopia, having been eliminated from Asia and some African countries. It has a significant socioeconomic impact because of the temporary disability that it causes. Dracunculiasis is exclusively caught from drinking water, usually from ponds. A campaign to eradicate the disease was launched in the 1980s and has made significant progress. The strategy of the campaign is discussed, including water supply, health education, case management, and vector control. Current issues including the integration of the campaign into primary health care and the mapping of cases by using geographic information systems are also considered. Finally, some lessons for other disease control and eradication programs are outlined.
Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4
Authors: Munson Erik L. , Du Chateau Brian K. , Jobe Dean A. , Lovrich Steven D. , Callister Steven M. , Schell Ronald F.
Published: July 2002

Borreliacidal antibody production is one of several parameters for establishing the effectiveness ofBorrelia burgdorferivaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages andB. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivatedB. burgdorferiin aluminum hydroxide, but not recombinant OspA. Anti-OspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.
Immunization with RecombinantPlasmodium yoeliiMerozoite Surface Protein 4/5 Protects Mice against Lethal Challenge
Authors: Kedzierski Lukasz , Black Casilda G. , Coppel Ross L.
Published: July 2002

Plasmodium yoeliimerozoite surface protein 4/5 (PyMSP4/5), expressed as a recombinant protein, was highly effective at protecting mice against lethal challenge withP. yoelii. There was a significant correlation between prechallenge antibody levels and peak parasitemia, suggesting that the homologues of PyMSP4/5 inPlasmodium falciparumare promising components of a subunit vaccine against malaria.
Released ATP Is an Extracellular Cytotoxic Mediator in Salivary Histatin 5-Induced Killing of
Candida albicans
Authors: Koshlukova Svetlana E. , Araujo Marcelo W. B. , Baev Didi , Edgerton Mira
Published: July 2002

Salivary histatins (Hsts) are antifungal peptides with promise as therapeutic agents against candidiasis. Hst 5 kills the fungal pathogen
Candida albicans
via a mechanism that involves release of cellular ATP in the absence of cytolysis. Here we demonstrate that released ATP has a further role in Hst 5 killing. Incubation of the cells with ATP analogues induced cell death, and addition of the ATP scavenger apyrase to remove extracellular ATP released during Hst 5 treatment resulted in a reduction in cell killing. Experiments using anaerobically grown
C. albicans
with decreased susceptibility to Hst 5 confirmed that depletion of cellular ATP as a result of ATP efflux was not sufficient to cause cell death. In contrast to Hst-susceptible aerobic cultures, anaerobically grown cells were not killed by exogenously applied ATP. These findings established that Hst binding, subsequent entry into the cells, and ATP release precede the signal for cytotoxicity, which is mediated by extracellular ATP. In a higher-eukaryote paradigm, released ATP acts as a cytotoxic mediator by binding to membrane nucleotide P2X receptors. Based on a pharmacological profile and detection of a
C. albicans
60-kDa membrane protein immunoreactive with antibody to P2X
7
receptor, we propose that released ATP in response to Hst 5 activates candidal P2X
7
-like receptors to cause cell death.
DNA Rearrangements in the Flagellin Locus of an
flaA
Mutant of
Campylobacter jejuni
during Colonization of Chicken Ceca
Authors: Nuijten Piet J. M. , van den Berg Anton J. G. , Formentini Ivan , van der Zeijst Bernard A. M. , Jacobs Anton A. C.
Published: July 2002

Campylobacter jejuni
is an enteropathogen for humans but commensal for chickens. In both hosts, the flagella and motility are important colonization factors. The flagellin gene is duplicated in
Campylobacter
, but only one flagellin gene,
flaA
, is sufficient for motility. We found that, during colonization of the chicken intestine, a nonmotile
flaA
mutant of
C. jejuni
underwent rearrangements within its flagellin locus, thereby regaining its motility and colonization capacity. In contrast, in vitro motile revertants isolated from liquid culture showed different flagellin DNA rearrangements than after reversion in the chicken.
Cloning and Characterization ofvuuA, a Gene Encoding theVibrio vulnificusFerric Vulnibactin Receptor
Authors: Webster Athena C. D. , Litwin Christine M.
Published: July 2002

The ability ofVibrio vulnificusto acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and inV. vulnificus, transcriptional regulation by iron depends on thefurgene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from aV. vulnificus furmutant had 53% homology with the first 15 amino acids of the mature protein of theVibrio choleraevibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor ofV. vulnificus. Analysis of the DNA sequence of thevuuApromoter region demonstrated a sequence identical to the upstream Fur box ofV. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence ofV. choleraeViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of theV. vulnificus vuuAgene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the completeviuAcoding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.
Biochemical and Immunological Characterization of MP65, a Major Mannoprotein Antigen of the Opportunistic Human Pathogen
Candida albicans
Authors: Gomez Maria J. , Maras Bruno , Barca Alessandra , La Valle Roberto , Barra Donatella , Cassone Antonio
Published: July 2002

In the search of the antigenic determinants of a 65-kDa mannoprotein (MP65) of
Candida albicans
, tryptic fragments of immunoaffinity-purified MP65 preparations were tested for their ability to induce lymphoproliferation of human peripheral blood mononuclear cells (PBMC). Five major peptides (T1 to T5) were shown to induce a vigorous proliferation of PBMC from the majority of the eight healthy human subjects tested. With the use of synthetic peptides, critical amino acid sequences of the two most immunoactive (T1 and T2) peptides were determined. Similar to what was found for the MP65 molecule, no PBMC multiplication was induced by the antigenic peptides in cultures of naive cord blood cells. The amino acid sequence analysis of tryptic and chymotryptic peptides of MP65 demonstrated a substantial homology with the deduced sequences of two cell wall proteins of
Saccharomyces cerevisiae
, encoded by the genes
YRM305C
and
YGR279C
. However, the antigenic peptides were those showing the least similarity with the corresponding regions of the above proteins. In particular, the lymphoproliferation-inducing sequence of the T1 peptide scored only 20% identity with the homologous regions of
S. cerevisiae
proteins. Besides disclosing the amino acid sequence of MP65, this study provides an initial characterization of some of its antigenic determinants, as well as of synthetic peptides of potential use to detect specific immune responses against MP65, a major target of anticandidal cell-mediated immunity in humans.
A Major Secreted Elastase Is Essential for Pathogenicity of
Aeromonas hydrophila
Authors: Cascón Alberto , Yugueros Javier , Temprano Alejandro , Sánchez María , Hernanz Carmen , Luengo José María , Naharro Germán
Published: July 2002

Aeromonas hydrophila
is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity,
ahyB
, was cloned from
Aeromonas hydrophila
AG2 into pUC18 and expressed in
Escherichia coli
and in the nonproteolytic species
Aeromonas salmonicida
subsp.
masoucida
. Nucleotide sequence analysis of the
ahyB
gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the
Pseudomonas aeruginosa
elastase (52% identity),
Helicobacter pylori
zinc metalloprotease (61% identity), or proteases from several species of
Vibrio
(52 to 53% identity). The gene
ahyB
was insertionally inactivated, and the construct was used to create an isogenic
ahyB
mutant of
A. hydrophila
. These first reports of a defined mutation in an extracellular protease of
A. hydrophila
demonstrate an important role in pathogenesis.
A Model for Sequestration of the Transmission Stages ofPlasmodium falciparum: Adhesion of Gametocyte-Infected Erythrocytes to Human Bone Marrow Cells
Authors: Rogers Nicola J. , Hall Belinda S. , Obiero Jacktone , Targett Geoffrey A. T. , Sutherland Colin J.
Published: July 2002

With the aim of developing an appropriate in vitro model of the sequestration of developingPlasmodium falciparumsexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays ofP. falciparumcytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.
Construction and Phenotypic Evaluation of aVibrio vulnificus vvpEMutant for Elastolytic Protease
Authors: Jeong Kwang Cheol , Jeong Hye Sook , Rhee Joon Haeng , Lee Shee Eun , Chung Sun Sik , Starks Angela M. , Escudero Gloria M. , Gulig Paul A. , Choi Sang Ho
Published: July 2002

Vibrio vulnificusis an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature ofV. vulnificusinfections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced thevvpEgene encoding an elastase ofV. vulnificusATCC 29307. The functions of the elastase were assessed by constructingvvpEinsertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed invvpEmutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinantvvpEgene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of theV. vulnificus vvpEgene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, avvpEmutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis ofV. vulnificusthan would have been predicted by examining the effects of administering purified proteins to animals. However,V. vulnificusutilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.
Induction of Interleukin-4 (IL-4) byLegionella pneumophilaInfection in BALB/c Mice and Regulation of Tumor Necrosis Factor Alpha, IL-6, and IL-1β
Authors: Newton Catherine , McHugh Shannon , Widen Ray , Nakachi Noriya , Klein Thomas , Friedman Herman
Published: July 2002

Infection of BALB/c mice with a sublethal concentration ofLegionella pneumophilacauses an acute disease that is resolved by innate immune responses. The infection also initiates the development of adaptive Th1 responses that protect the mice from challenge infections. To study the early responses, cytokines induced during the first 24 h after infection were examined. In the serum, interleukin-12 (IL-12) was detectable by 3 h and peaked at 10 h, while gamma interferon was discernible by 5 h and peaked at 8 h. Similar patterns were observed in ex vivo cultures of splenocytes. A transient IL-4 response was also detected by 3 h postinfection in ex vivo cultures. BALB/c IL-4-deficient mice were more susceptible toL. pneumophilainfection than were wild-type mice. The infection induced higher serum levels of acute-phase cytokines (tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6), and reducing TNF-α levels with antibodies protected the mice from death. Moreover, the addition of IL-4 toL. pneumophila-infected macrophage cultures suppressed the production of these cytokines. Thus, the lack of IL-4 in the deficient mice resulted in unchecked TNF-α production, which appeared to cause the mortality. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is induced by IL-4 duringListeria monocytogenesinfection, was detected at between 2 and 30 h after infection. However, MCP-1 did not appear to be induced by IL-4 or to be required for the TNF-α regulation by IL-4. The data suggest that the early increase in IL-4 serves to regulate the mobilization of acute phase cytokines and thus controls the potential harmful effects of these cytokines.
Restriction ofToxoplasma gondiiGrowth in Human Brain Microvascular Endothelial Cells by Activation of Indoleamine 2,3-Dioxygenase
Authors: Däubener Walter , Spors Birgit , Hucke Christian , Adam Rüdiger , Stins Monique , Kim Kwang Sik , Schroten Horst
Published: July 2002

One of the first steps in the development of cerebral toxoplasmosis is the penetration of the blood-brain barrier, which is comprised of microvascular endothelial cells. We examined the capacity of human brain microvascular endothelial cells (HBMEC) to interact withToxoplasma gondii. We found that stimulation of HBMEC with gamma interferon (IFN-γ) resulted in the induction of toxoplasmostasis. The capacity of HBMEC to restrictToxoplasmagrowth after IFN-γ stimulation was enhanced in the presence of tumor necrosis factor alpha (TNF-α). In addition, we found that IFN-γ induced a strong induction of indoleamine 2,3-dioxygenase (IDO) activity in HBMEC, and this enzyme activity was enhanced by costimulation with TNF-α. The addition of excess amounts of tryptophan to the HBMEC cultures resulted in a complete abrogation of the IFN-γ–TNF-α-mediated toxoplasmostasis. We therefore conclude that IDO induction contributed to the antiparasitic effector mechanism inducible in HBMEC by IFN-γ and TNF-α.
Enteropathogenic
Escherichia
coli
Virulence Factor Bundle-Forming Pilus Has a Binding Specificity for Phosphatidylethanolamine
Authors: Khursigara C. , Abul-Milh M. , Lau B. , Girón J. A. , Lingwood C. A. , Foster D. E. Barnett
Published: July 2002

The bundle-forming pilus (BFP) of enteropathogenic
Escherichia
coli
(EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a
bfp
-carrying plasmid. Strains which did not express BFP did not bind PE, including a
bfpA
disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP
-
negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.
Analysis of Cytokines in the Early Development of Gastric Secondary Lymphoid Follicles inHelicobacter pylori-Infected BALB/c Mice with Neonatal Thymectomy
Authors: Uchida Kazushige , Okazaki Kazuichi , Debrecceni Andras , Nishi Toshiki , Iwano Hirosi , Inai Maki , Uose Suguru , Nakase Hiroshi , Ohana Masaya , Oshima Chikashi , Matsushima Yumi , Kawanami Chiharu , Hiai Hiroshi , Masuda Toru , Chiba Tsutomu
Published: July 2002

Immunological interaction between the host andHelicobacter pyloriseems to play a critical role in follicular formation in gastric mucosa. We reportedH. pylori-induced follicular gastritis model using neonatally thymectomized mice. In this study, we investigated the involvement of various cytokines in this model. BALB/c mice were thymectomized on the third day after birth (nTx). At 6 weeks old, these mice were orally infected withH. pylori. Histological studies showed that follicular formation occurred from 8 weeks after the infection and that most of the infiltrating lymphocytes were CD4+and B cells. Neutrophils increased transiently at 1 week after the infection. Gamma interferon, interleukin-7 (IL-7), and IL-7 receptor were expressed in the stomach of the nTx mice irrespective of the infection. In contrast, expressions of the tumor necrosis factor alpha, IL-4 and lymphotoxin-α genes were remarkably upregulated by the infection. Our findings suggest that follicular formation may require cooperative involvement of a Th2-type immune response, tumor necrosis factor alpha and lymphotoxin-α in addition to the Th1-type immune response inH. pylori-induced gastritis in nTx mice.
Role for Inducible Nitric Oxide Synthase in Protection from ChronicChlamydia trachomatisUrogenital Disease in Mice and Its Regulation by Oxygen Free Radicals
Authors: Ramsey K. H. , Sigar I. M. , Rana S. V. , Gupta J. , Holland S. M. , Byrne G. I.
Published: July 2002

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2−/−) mice resolveChlamydia trachomatisgenital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47phox−/−) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT)NOS2+/+andNOS2−/−controls. When we induced an in vivo chemical block in iNOS activity in p47phox−/−mice usingNG-monomethyl-l-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47phox−/−controls, L-NMMA-treated p47phox−/−mice resolved their infections more rapidly. However, L-NMMA-treated p47phox−/−mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that forNOS2−/−mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.
Complete Nucleotide Sequence of a
Staphylococcus aureus
Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C
Authors: Yamaguchi Takayuki , Hayashi Tetsuya , Takami Hideto , Ohnishi Makoto , Murata Takahiro , Nakayama Keisuke , Asakawa Kayo , Ohara Masaru , Komatsuzawa Hitoshi , Sugai Motoyuki
Published: July 2002

The complete nucleotide sequence of pETB, a 38.2-kb
Staphylococcus aureus
plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS
257
, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the
etb
gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of
etb
-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.
Blockade of Caspases Inhibits Amebic Liver Abscess Formation in a Mouse Model of Disease
Authors: Yan Le , Stanley Samuel L.
Published: July 2002

We looked at the effect of inhibiting caspases on amebic liver abscess in the mouse model of infection. A dose of the pan-caspase inhibitor benzyloxycarbonyl-V-A-D-O-methyl fluoromethyl ketone (Z-VAD-FMK; R & D Systems) given to SCID mice 2 h prior to direct hepatic inoculation with
Entamoeba histolytica
trophozoites, and 12 h after amebic inoculation, reduced the mean liver abscess size by 70% at 24 h compared to a control group. These data indicate that apoptosis plays a significant but not an exclusive role in amebic liver abscess formation in the mouse model.