bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of P55, a Multidrug Efflux Pump in
Mycobacterium bovis
and
Mycobacterium tuberculosis
Authors: Silva Pedro E. A. , Bigi Fabiana , de la Paz Santangelo Marı́a , Romano Maria Isabel , Martı́n Carlos , Cataldi Angel , Aı́nsa José A.
Published: July 2002

The
Mycobacterium bovis P55
gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of
Mycobacterium tuberculosis
H37Rv, annotated as a probable drug efflux protein. Genes similar to
P55
were present in all species of the
M. tuberculosis
complex and other mycobacteria such as
Mycobacterium leprae
and
Mycobacterium avium
. By Western blotting, P55 was located in the membrane fraction of
M. bovis
. When transformed into
Mycobacterium smegmatis
after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide
m
-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine.
M. smegmatis
cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.
DNA Transformation of
Leishmania infantum
Axenic Amastigotes and Their Use in Drug Screening
Authors: Sereno Denis , Roy Gaétan , Lemesre Jean Loup , Papadopoulou Barbara , Ouellette Marc
Published: July 2002

Protocols for DNA electroporation in
Leishmania
promastigote cells are well established. More recently, in vitro culture of axenic
Leishmania
amastigotes became possible. We have established conditions for DNA transformation of axenically grown
Leishmania infantum
amastigotes. Parameters for DNA electroporation of
Leishmania
axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 μM (0.12 μg/ml), 55 μg/ml, 95 μg/ml, and 0.12 μg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.
Human Malaria in Immunocompromised Mice: New In Vivo Model for Chemotherapy Studies
Authors: Moreno A. , Badell E. , Van Rooijen N. , Druilhe P.
Published: July 2002

We have recently designed a new
Plasmodium falciparum
mouse model and documented its potential for the study of immune effector mechanisms. In order to determine its value for drug studies, we evaluated its response to existing antimalarial drugs compared to that observed in humans. Immunocompromised BXN (
bg/bg xid/xid nu/nu
) mice were infected with either the sensitive NF54 strain or the multiresistant T24 strain and then treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. A parallelism was observed between previously reported human responses and
P. falciparum
-parasitized human red blood cell (huRBC)–BXN mouse responses to classical antimalarial drugs, measured in terms of speed of decrease in parasitemia and of morphological alterations of the parasites. Mice infected with the sensitive strain were successfully cured after treatment with either chloroquine or mefloquine. In contrast, mice infected with the multiresistant strain failed to be cured by chloroquine or quinine but thereafter responded to dihydroartemisinin treatment. The speed of parasite clearance and the morphological alterations induced differed for each drug and matched previously reported observations, hence stressing the relevance of the model. These data thus suggest that
P. falciparum
-huRBC–BXN mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of
P. falciparum
responses to drugs.
Differential C Isotope Discrimination by Fungi during Decomposition of C3- and C4-Derived Sucrose
Authors: Henn Matthew R. , Chapela Ignacio H.
Published: July 2002

Stable isotope analysis is a major tool used in ecosystem studies to establish pathways and rates of C exchange between various ecosystem components. Little is known about isotopic effects of many such components, especially microbes. Here we report on the discovery of an unexpected pattern of C isotopic discrimination by basidiomycete fungi with far-reaching consequences for our understanding of isotopic processing in ecosystems where these microbes mediate material transfers across trophic levels. We measured fractionation effects on three ecologically relevant basidiomycete species under controlled laboratory conditions. Sucrose derived from C3and C4plants is fractionated differentially by these microbes in a taxon-specific manner. The differentiation between mycorrhizal and saprotrophic fungi observed in the field by others is not explained by intrinsic discrimination patterns. Fractionation occurs during sugar uptake and is sensitive to the nonrandom distribution of stable isotopes in the sucrose molecule. The balance between respiratory physiology and fermentative physiology modulates the degree of fractionation. These discoveries disprove the assumption that fungal C processing does not significantly alter the distribution of stable C isotopes and provide the basis for a reevaluation of ecosystem models based on isotopic evidence that involve C transfer across microbial interfaces. We provide a mechanism to account for the observed differential discrimination effects.
Biochemical and Genetic Characterization of Propionicin T1, a New Bacteriocin from
Propionibacterium thoenii
Authors: Faye Therese , Langsrud Thor , Nes Ingolf F. , Holo Helge
Published: July 2002

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of
Propionibacterium thoenii
. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of
Propionibacterium acidipropionici
,
Propionibacterium thoenii
, and
Propionibacterium jensenii
tested and also against
Lactobacillus sake
NCDO 2714 but showed no activity against
Propionibacterium freudenreichii
. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene
pctA
was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical
sec
leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of
pctA
. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.
Inhibitory Effects of Collagen on the PCR for Detection of
Clostridium perfringens
Authors: Kim Sangburm , Labbe Ronald G. , Ryu Sangryeol
Published: July 2002

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic
Clostridium perfringens
by PCR with respect to the kind of food being analyzed.
Rapid Methods for Testing the Efficacy of Sterilization-Grade Filter Membranes
Authors: Griffiths Matthew H. , Andrew Peter W. , Ball Peter R. , Hall George M.
Published: July 2002

The validation of sterilization-grade membranes is integral to ensuring the efficient and safe use of microfiltration systems. Here validation refers to the production of sterile filtrate for sterilizing-grade membranes under challenge test conditions. Current validation methods require 48 h of culture for results to become available, which creates time delays within the manufacturing process and quality control (QC) backlogs. This work compares four methods for the production of filter challenge test data, to the desired test sensitivity, within 24 h using bioluminescent and fluorescent recombinant strains of the test organism
Brevundimonas diminuta
. These methods should provide a way to implement more rapid QC test regimens for filters.
Cloning of the
spoT
Gene of “
Candidatus
Phlomobacter fragariae” and Development of a PCR-Restriction Fragment Length Polymorphism Assay for Detection of the Bacterium in Insects
Authors: Foissac Xavier , Danet Jean-Luc , Zreik Leyla , Gandar Jeanne , Nourrisseau Jean-Georges , Bové Joseph-Marie , Garnier Monique
Published: July 2002

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium “
Candidatus
Phlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between “P. fragariae” and other insect-associated proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” open reading frame sharing appreciable homology with the
spoT
gene from other proteobacteria. A
spoT
-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish “P. fragariae” from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry “P. fragariae.” Interestingly however, the “P. fragariae”
spoT
sequence could be easily detected in whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.
Isolation of Mycobacteria from Frozen Fish Destined for Human Consumption
Authors: Mediel M. J. , Rodriguez V. , Codina G. , Martin-Casabona N.
Published: July 2002

Mycobacteria were isolated from defrost water and tissue of sole (
Solea solea
), hake (
Merluccius merluccius
), cod (
Gadus morhua
), ling (
Genypterus blacodes
), and monkfish (
Lophius piscatorius
) on Löwenstein-Jensen medium after incubation at different temperatures. Samples of frozen fish were obtained under sterile conditions inside a refrigeration chamber (−18 to −22°C) in a wholesale market from which these products are distributed to shops for retail sale and human consumption.
Metabolic Engineering of
Lactobacillus helveticus
CNRZ32 for Production of Pure
l
-(+)-Lactic Acid
Authors: Kylä-Nikkilä Kari , Hujanen Mervi , Leisola Matti , Palva Airi
Published: July 2002

Expression of
d
-(−)-lactate dehydrogenase (
d
-LDH) and
l
-(+)-LDH genes (
ldhD
and
ldhL
, respectively) and production of
d
-(−)- and
l
-(+)-lactic acid were studied in
Lactobacillus helveticus
CNRZ32. In order to develop a host for production of pure
l
-(+)-isomer of lactic acid, two
ldhD
-negative
L. helveticus
CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the
ldhD
gene, and the other was constructed by replacing the structural gene of
ldhD
with an additional copy of the structural gene (
ldhL
) of
l
-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the
ldhL
structural gene was expressed under the
ldhD
promoter. The two
d
-LDH-negative strains produced only
l
-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum
l
-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for
l
-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41°C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.
Role of the DmpR-Mediated Regulatory Circuit in Bacterial Biodegradation Properties in Methylphenol-Amended Soils
Authors: Sarand Inga , Skärfstad Eleonore , Forsman Mats , Romantschuk Martin , Shingler Victoria
Published: July 2002

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the
dmp
operon genes encoding the (methyl)phenol degradative pathway of
Pseudomonas
sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to
para
-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.
Relative Distribution and Conservation of Genes Encoding Aminoglycoside-Modifying Enzymes in
Salmonella enterica
Serotype Typhimurium Phage Type DT104
Authors: Frana Timothy S. , Carlson Steve A. , Griffith Ronald W.
Published: July 2002

PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 422 veterinary isolates of
Salmonella enterica
serotype Typhimurium. The identities of extra-integron genes encoding resistance to streptomycin, gentamicin, kanamycin, and apramycin were evaluated. Gentamicin resistance was conferred by the
aadB
gene. Kanamycin resistance was encoded by either the
aphA1-Iab
gene or the
Kn
gene. Apramycin resistance was determined by the
aacC4
gene. Analysis of gene distribution did not reveal significant differences with regard to phage type, host species, or region except for the
Kn
gene, which was found mostly in nonclinical isolates. The data from this study indicate that pentaresistant DT104 does not acquire extra-integron genes in species- or geography-related foci, which supports the hypothesis that clonal expansion is the method of spread of this organism.
Solid-Phase Capture of Proteins, Spores, and Bacteria
Authors: Weimer B. C. , Walsh M. K. , Beer C. , Koka R. , Wang X.
Published: July 2002

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-
Escherichia coli
O157:H7, -
Bacillus globigii
, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 μg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-
B. globigii
and anti-
E. coli
O157:H7 modified beads. The lower limit of detection for both
E. coli
O157:H7 and
B. globigii
was 1 spore/cell independent of the sample size. The activity of anti-
B. globigii
modified beads declined after 3 days. Anti-
E. coli
O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25°C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.
Rupture of the Cell Envelope by Decompression of the Deep-Sea Methanogen
Methanococcus jannaschii
Authors: Park Chan Beum , Clark Douglas S.
Published: July 2002

The effect of decompression on the structure of
Methanococcus jannaschii
, an extremely thermophilic deep-sea methanogen, was studied in a novel high-pressure, high-temperature bioreactor. The cell envelope of
M. jannaschii
appeared to rupture upon rapid decompression (ca. 1 s) from 260 atm of hyperbaric pressure. When decompression from 260 atm was performed over 5 min, the proportion of ruptured cells decreased significantly. In contrast to the effect produced by decompression from hyperbaric pressure, decompression from a hydrostatic pressure of 260 atm did not induce cell lysis.
Genes from
Pseudomonas
sp. Strain BS Involved in the Conversion of
l
-2-Amino-Δ
2
-Thiazolin-4-Carbonic Acid to
l
-Cysteine
Authors: Shiba Toshikazu , Takeda Kohji , Yajima Misako , Tadano Makoto
Published: July 2002

dl
-2-amino-Δ
2
-thiazolin-4-carbonic acid (
dl
-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of
l
-ATC to
l
-cysteine from
Pseudomonas
sp. strain BS. The introduction of this DNA fragment into
Escherichia coli
cells enabled them to convert
l
-ATC to cysteine via
N
-carbamyl-
l
-cysteine (
l
-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has
l
-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the
l
-cysteine synthesis from
dl
-ATC. These ORFs were designated
atcB
and
atcC
, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of
E. coli
cells carrying deletion derivatives of pTK10. The results indicate that
atcB
and
atcC
are involved in the conversion of
l
-ATC to
l
-NCC and the conversion of
l
-NCC to cysteine, respectively.
atcB
was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with
l
-
N
-carbamoylases. Since both enzymes can only catalyze the
l
-specific conversion from
l
-ATC to
l
-NCC or
l
-NCC to
l
-cysteine, it is thought that
atcB
and
atcC
encode
l
-ATC hydrolase and
N
-carbamyl-
l
-cysteine amidohydrolase, respectively.
Characterization of
Trichodesmium
spp. by Genetic Techniques
Authors: Orcutt K. M. , Rasmussen U. , Webb E. A. , Waterbury J. B. , Gundersen K. , Bergman B.
Published: July 2002

The genetic diversity of
Trichodesmium
spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a
hetR
fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of
Trichodesmium
spp. from the two hemispheres. Culture isolates of
Trichodesmium thiebautii
,
Trichodesmium hildebrandtii
,
Trichodesmium tenue
, and
Katagnymene spiralis
displayed remarkable similarity when these techniques were used, suggesting that
K. spiralis
is very closely related to the genus
Trichodesmium
. The largest genetic variation was found between
Trichodesmium erythraeum
and all other species of
Trichodesmium
, including a species of
Katagnymene
. Our data obtained with all three techniques suggest that there are two major clades of
Trichodesmium
spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.
Superantigens and Cystic Fibrosis: Resistance of Presenting Cells to Dexamethasone
Authors: Ben-Ari Josef , Gozal David , Dorio Raymond J. , Bowman C. Michael , Reiff Andreas , Walker Sharyn M.
Published: July 2002

Staphylococcus aureus, a common pulmonary pathogen in cystic fibrosis (CF), produces exotoxins that are extremely potent superantigens. A number of animal studies have shown that superantigens cause pulmonary inflammation, but the possible role of superantigens in CF has not been investigated. The present study assessed possible differences between control and CF B cells in presenting superantigens to T cells. Immortalized B-cell lines were used as superantigen-presenting cells to avoid environmental influences (e.g., infection or antibiotics) common to freshly isolated cells. The results show that CF B-cell lines presented a staphylococcal superantigen to the immortalized T-cell line (Jurkat) as effectively as did control B-cell lines as measured by interleukin-2 production. However, in contrast to the case for control B-cell lines, dexamethasone did not inhibit CF B-cell lines from presenting superantigen. The resistance of superantigen-presenting CF B cells to corticosteroids suggests that the pulmonary response to superantigens may be poorly regulated in CF, leading to an exaggerated inflammatory response toS. aureus.
Serum Opsonic Activity in Infants with Sickle-Cell Disease Immunized with Pneumococcal Polysaccharide Protein Conjugate Vaccine
Authors: Nowak-Wegrzyn Anna , Winkelstein Jerry A. , Swift Andrea J. , Lederman Howard M. ,   The Pneumococcal Conjugate Vaccine Study Group
Published: July 2002

Pneumococcal infections are an important cause of morbidity and mortality in children with sickle-cell disease (SCD). Pneumococcal conjugate vaccines (PCVs) are immunogenic in healthy infants <2 years of age but have not been evaluated in young children with SCD. Infants with SCD were immunized with a 7-valent PCV (Wyeth-Lederle Vaccines & Pediatrics) at 2, 4, and 6 months of age. A booster dose of 23-valent pneumococcal polysaccharide vaccine (PPV; Pnu-Immune) was administered at 24 months of age. Antipneumococcal type 6B and 14 serum opsonic activity was measured to assess the biologic function of the antibody. Following the administration of three doses of PCV, opsonic activity against serotype 6B increased from 4.8% at 2 months to 33.5% at 7 months, with a subsequent decline to 8.1% at 12 months and 7.5% at 24 months and with an increase to 30.7% at 25 months after administration of a booster dose of PPV. Similar trends were seen with serotype 14 (opsonic activities were 9.4% at 2 months, 24.9% at 7 months, 16.5% at 12 months, and 12.6% at 24 months, and the opsonic activity was 27.3% 1 month after the administration of PPV). Serum opsonic activity correlated with antibody levels for both serotypes. PCV induces serum opsonic activity in infants with SCD. Antipneumococcal serum opsonic activity correlates with antibody levels.
Performance of Antigens Used in Detecting Delayed-Type Hypersensitivity in Adolescents Infected with the Human Immunodeficiency Virus
Authors: Rogers Audrey Smith , Ellenberg Jonas H. , Douglas Steven D. , Henry-Reid Lisa , Peralta Ligia , Wilson Craig M. ,   The Adolescent Medicine HIV/AIDS Research Network
Published: July 2002

We examined the performance of delayed-type hypersensitivity (DTH) antigens employing a new
Candida albicans
product in a human immunodeficiency virus (HIV)-infected and nonanergic adolescent population. Diameters of induration (in millimeters) for three intradermally applied antigens (
C. albicans
, tetanus toxoid, and mumps) were compared in a population of HIV-infected 12 to 18 year olds at study entry in a national multicenter study of HIV disease progression. CD4
+
T-cell counts were measured in quality-controlled laboratories. The influence of past immunization, gender, and clinical status on antigen reactivity was evaluated with contingency table comparisons and relative risk estimation. Nearly one-half of the 123 eligible subjects were untreated, and almost three-quarters were early in HIV disease by clinical indicators. There was no statistically significant difference in reactivity by past immunization status. Candida antigen (CASTA; Greer Laboratories) evoked DTH response in a significantly higher number of males and females at every level of induration (largest
P
value, 0.049 for male comparisons; all
P
values, <0.001 for females) and in subjects with early and intermediate HIV disease at every level of induration (all
P
values, <0.0001) than either tetanus or mumps antigens. No two-antigen combination was as useful as all three antigens across either gender or clinical categories, although candida and tetanus was the most useful two-antigen combination at indurations of <3 mm. The superior performance of a new C.
albicans
antigen may extend the utility of DTH assessment in monitoring immune function.
Immunoblot Profile as Predictor of Toxoplasmic Encephalitis in Patients Infected with Human Immunodeficiency Virus
Authors: Leport Catherine , Franck Jacqueline , Chene Genevieve , Derouin Francis , Ecobichon Jean-Luc , Pueyo Sophie , Miro Jose M. , Luft Benjamin J. , Morlat Philippe , Dumon Henri ,   the ANRS 005– ACTG 154 Study Group
Published: July 2002

In order to define more accurately human immunodeficiency virus-infected patients at risk of developing toxoplasmic encephalitis (TE), we assessed the prognostic significance of the anti-
Toxoplasma gondii
immunoglobulin G (IgG) immunoblot profile, in addition to AIDS stage, a CD4
+
cell count <50/mm
3
, and an antibody titer ≥150 IU/ml, in patients with CD4 cell counts <200/mm
3
and seropositive for
T. gondii.
Baseline serum samples from 152 patients included in the placebo arm of the ANRS 005-ACTG 154 trial (pyrimethamine versus placebo) were used. The IgG immunoblot profile was determined using a
Toxoplasma
lysate and read using the Kodak Digital Science 1D image analysis software. Mean follow-up was 15.1 months, and the 1-year incidence of TE was 15.9%. The cumulative probability of TE varied according to the type and number of anti-
T. gondii
IgG bands and reached 65% at 12 months for patients with IgG bands of 25 and 22 kDa. In a Cox model adjusted for age, gender, Centers for Disease Control and Prevention (CDC) clinical stage, and CD4 and CD8 cell counts, the incidence of TE was higher when the IgG 22-kDa band (hazard ratio [HR] = 5.4;
P
< 0.001), the IgG 25-kDa band (HR = 4.7;
P
< 0.001), or the IgG 69-kDa band (HR = 3.4;
P
< 0.001) was present and was higher for patients at CDC stage C (HR = 4.9;
P
< 0.001).
T. gondii
antibody titer and CD4 cell count were not predictive of TE. Thus, detection of IgG bands of 25, 22, and/or 69 kDa may be helpful for deciding when primary prophylaxis for TE should be started or discontinued, especially in the era of highly active antiretroviral therapy.
Expression of Interleukin-9 Leads to Th2 Cytokine-Dominated Responses and Fatal Enteropathy in Mice with ChronicSchistosoma mansoniInfections
Authors: Fallon Padraic G. , Smith Philip , Richardson Emma J. , Jones Frances J. , Faulkner Helen C. , Van Snick Jacques , Renauld Jean-Christophe , Grencis Richard K. , Dunne David W.
Published: July 2002

Mice infected withSchistosoma mansonidevelop Th2 cytokine-mediated granulomatous pathology that is focused on the liver and intestines. In this study, transgenic mice constitutively expressing IL-9 were infected withS. mansoniand the outcome of infection was determined. Eight weeks after infection, transgenic mice with acute infections had a moderate increase in Th2 cytokine production but were overtly normal with respect to parasite infection and pathological responses. Transgenic mice with chronic infections died 10 weeks after infection, with 86% of transgenic mice dead by week 12 of infection, compared to 7% mortality in infected wild-type mice. Stimulation of mesenteric lymph node cells from infected transgenic mice with parasite antigen elicited elevated interleukin-4 (IL-4) and IL-5 production and reduced gamma interferon and tumor necrosis factor alpha production compared to the responses in wild-type mice. Morbid transgenic mice had substantial enlargement of the ileum, which was associated with muscular hypertrophy, mastocytosis, eosinophilia, goblet cell hyperplasia, and increased mucin expression. We also observed that uninfected transgenic mice exhibited alterations in their intestines. Although there was hepatic mastocytosis and eosinophilia in infected transgenic mice, there was no hepatocyte damage. Death of transgenic mice expressing IL-9 during schistosome infection was primarily associated with enteropathy. This study highlights the pleiotropic in vivo activity of IL-9 and demonstrates that an elevated Th2 cytokine phenotype leads to death during murine schistosome infection.
Human Granulocytic Ehrlichiosis Agent Inhibits Superoxide Anion Generation by Human Neutrophils
Authors: Mott Jason , Rikihisa Yasuko
Published: July 2002

The human granulocytic ehrlichiosis (HGE) agent, which replicates in neutrophils, was found not to induce superoxide anion (O2−) generation or extracellular release by human peripheral blood neutrophils, as measured by a luminol-dependent chemiluminescence assay or a cytochromecreduction assay, respectively. Furthermore, the HGE agent completely prevented O2−release by neutrophils upon stimulation with phorbol myristate acetate (PMA), formylmethionyl-leucyl-phenylalanine, orEscherichia coli. The inhibition was HGE agent dose dependent, required ehrlichial contact with the host cells, and was reversible upon removal of the extracellular HGE agent bound to the host cells prior to PMA stimulation. Structural integrity of or new protein synthesis by the HGE agent was not required for the inhibition; carbohydrate but not surface protein of the HGE agent was required. The HGE agent did not prevent O2−generation in human peripheral blood monocytes derived from the same individual. This neutrophil-specific prevention of O2−generation by the HGE agent would be critical in survival of the HGE agent. This is the first demonstration of the rapid inhibition of preexisting NADPH oxidase in human neutrophils by the HGE agent.
Pathogenic Mechanism of Mouse Brain Damage Caused by Oral Infection with Shiga Toxin-ProducingEscherichia coliO157:H7
Authors: Kita Eiji , Yunou Yoshihisa , Kurioka Takaaki , Harada Hiroko , Yoshikawa Shinji , Mikasa Keiichi , Higashi Nobutaka
Published: July 2002

In a previous study, we showed that infection with Shiga toxin (Stx)-producingEscherichia coliO157:H7 (strain SmrN-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain SmrN-9 elicited a tumor necrosis factor alpha (TNF-α) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-α was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-α (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-α alone or TNF-α and Stx2. In contrast, primary exposure of HUVEC to TNF-α followed by exposure to Stx2 alone or TNF-α and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-α after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-α. Such cell injury may be a prerequisite to brain damage after infection with Stx-producingE. coliO157:H7.
Identification of
Brucella suis
Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis
Authors: Foulongne Vincent , Bourg Giséle , Cazevieille Chantal , Michaux-Charachon Sylvie , O'Callaghan David
Published: July 2002

Bacteria of the genus
Brucella
are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of
Brucella
virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152
Brucella suis
1330 tagged mini-Tn
5
Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D
3
-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the
virB
operon and
manB
), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of
Brucella
for the different factors identified are discussed.
Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody
Authors: Han Yongmoon , Riesselman Marcia H. , Cutler Jim E.
Published: July 2002

We previously reported that a liposome-mannan vaccine (L-mann) ofCandida albicansinduces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for aC. albicanscell surface β-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6.1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginalCandidaCFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.
Effects of Iron on Extracellular and Intracellular Growth of
Penicillium marneffei
Authors: Taramelli D. , Brambilla S. , Sala G. , Bruccoleri A. , Tognazioli C. , Riviera-Uzielli L. , Boelaert J. R.
Published: July 2002

Killing of intracellular
Penicillium marneffei
conidia is demonstrated in gamma interferon-lipopolysaccharide-activated human THP1 and mouse J774 cells. Iron overload significantly reduces the antifungal activity of macrophages. Likewise, exogenous iron enhances and iron chelators inhibit the extracellular growth of
P. marneffei
. These results suggest that iron availability critically affects immunity to and the pathogenicity of
P. marneffei
.
The
sigA
Gene Which Is Borne on the
she
Pathogenicity Island of
Shigella flexneri
2a Encodes an Exported Cytopathic Protease Involved in Intestinal Fluid Accumulation
Authors: Al-Hasani Keith , Henderson Ian R. , Sakellaris Harry , Rajakumar Kumar , Grant Travis , Nataro James P. , Robins-Browne Roy , Adler Ben
Published: July 2002

In this study, the
sigA
gene situated on the
she
pathogenicity island of
Shigella flexneri
2a was cloned and characterized. Sequence analysis showed that
sigA
encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of
Enterobacteriaceae
) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of
Shigella
infections. SigA was at least partly responsible for the ability of
S. flexneri
to stimulate fluid accumulation in ligated rabbit ileal loops.
The Flagellar Sigma Factor FliA (ς28) Regulates the Expression ofSalmonellaGenes Associated with the Centisome 63 Type III Secretion System
Authors: Eichelberg Katrin , Galán Jorge E.
Published: July 2002

One of the essential features of all pathogenic strains ofSalmonella entericais the ability to enter into nonphagocytic cells. This pathogenic property is mediated by theSalmonellapathogenicity island 1 (SPI-1)-encoded type III secretion system. Expression of components and substrates of this system is subject to complex regulatory mechanisms. These mechanisms include a number of specific and global transcriptional regulatory proteins. In this study we have compared inS. entericaserovars Typhimurium and Typhi the effect of mutations in flagellar genes on the phenotypes associated with the SPI-1 type III protein secretion system. We found that serovar Typhi strains carrying a null mutation in either of the flagellar regulatory genesflhDCorfliAwere severely deficient in entry into cultured epithelial cells and macrophage cytotoxicity. This defect could not be reversed by applying a mild centrifugal force, suggesting that the effects of the mutations were not due to the absence of motility. In contrast, the same mutations had no significant effect on the ability of serovar Typhimurium to enter into cultured Henle-407 cells or to induce macrophage cell death. Consistent with these observations, we found that the mutations in the flagellar regulatory proteins significantly reduced the expression of components of the SPI-1-encoded type III system in serovar Typhi but had a marginal effect in serovar Typhimurium. Our results therefore indicate that there is an overlap between regulatory mechanisms that control flagellar and type III secretion gene expression inSalmonellaserovar Typhi.
Listeriolysin O as a Reporter To Identify Constitutive and In Vivo-Inducible Promoters in the Pathogen
Listeria monocytogenes
Authors: Dubail Iharilalao , Berche Patrick ,   The European Listeria Genome Consortium, Charbit Alain
Published: July 2002

Listeria monocytogenes
is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by
hly
. LLO-negative mutants of
L. monocytogenes
are avirulent in the mouse model. We have developed a genetic system with
hly
as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting
L. monocytogenes
chromosomal fragments upstream of the promoterless
hly
gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this
hly
-based promoter trap system, combined with access to the
L. monocytogenes
genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the
hly
-based system as an in vivo expression technology tool, nine in vivo-induced loci of
L. monocytogenes
were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative
N
-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of
hly
as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in
L. monocytogenes
.
Characterization of the
Streptococcus mutans
Pyruvate Formate-Lyase (PFL)-Activating Enzyme Gene by Complementary Reconstitution of the In Vitro PFL-Reactivating System
Authors: Yamamoto Yasuhito , Sato Yutaka , Takahashi-Abbe Shoko , Takahashi Nobuhiro , Kizaki Harutoshi
Published: July 2002

The
act
gene was identified and an
act
mutant as well as the
pfl
mutant was constructed in
Streptococcus mutans
. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The
S. mutans act
gene encoded the sole enzyme able to activate the PFL protein in this organism.
Shiga Toxin-Induced Tumor Necrosis Factor Alpha Expression: Requirement for Toxin Enzymatic Activity and Monocyte Protein Kinase C and Protein Tyrosine Kinases
Authors: Foster Gregory H. , Armstrong Cassandra S. , Sakiri Ramesh , Tesh Vernon L.
Published: July 2002

Infections with Shiga toxin (Stx)-producing bacteria cause bloody diarrhea which may progress to life-threatening complications, including acute renal failure and neurological abnormalities. The precise mechanism of disease progression is unclear, although evidence suggests that the localized production of the host proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 may exacerbate toxin-mediated vascular damage. Purified Stxs have been demonstrated to elicit proinflammatory cytokine synthesis from human peripheral blood mononuclear cells and monocytic cell lines in vitro. To understand toxin-monocyte interactions required for cytokine synthesis, we have treated differentiated THP-1 cells with purified wild-type toxins, enzymatic mutants, or B subunits and measured TNF-α production. Our data suggest that A subunit enzymatic activity is essential for cytokine production. THP-1 cells were treated with a series of protein kinase C (PKC), PKA, and protein tyrosine kinase inhibitors to examine the role of intracellular signaling molecules in Stx-mediated cytokine production. Treatment of cells with PKC and tyrosine kinase inhibitors blocked TNF-α secretion by Stx-stimulated THP-1 cells. Stx treatment directly activated PKC, which occurred at a point upstream of transcriptional activation of the gene encoding TNF-α.
Susceptibility to SecondaryFrancisella tularensisLive Vaccine Strain Infection in B-Cell-Deficient Mice Is Associated with Neutrophilia but Not with Defects in Specific T-Cell-Mediated Immunity
Authors: Bosio Catharine M. , Elkins Karen L.
Published: July 2002

Previous studies have demonstrated a role for B cells, not associated with antibody production, in protection against lethal secondary infection of mice withFrancisella tularensislive vaccine strain (LVS). However, the mechanism by which B cells contribute to this protection is not known. To study the specific role of B cells during secondary LVS infection, we developed an in vitro culture system that mimics many of the same characteristics of in vivo infection. Using this culture system, we showed that B cells do not directly control LVS infection but that control of LVS growth is mediated primarily by LVS-primed T cells. Importantly, B cells were not required for the generation of effective memory T cells since LVS-primed, B-cell-deficient (BKO) mice generated CD4+and CD8+T cells that controlled LVS infection similarly to LVS-primed CD4+and CD8+T cells from wild-type mice. The control of LVS growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and BKO mice. Rather, the inability of BKO mice to survive secondary LVS infection was associated with marked neutrophil influx into the spleen very early after challenge. The neutrophilia was directly associated with B cells, since BKO mice reconstituted with naive B cells prior to a secondary challenge with LVS had decreased bacterial loads and neutrophils in the spleen and survived.