bluuebunny/crossref_metadata_2025 huggingface dataset:
Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in
Aureobasidium pullulans
Authors: Webb Jeremy S. , Barratt Sarah R. , Sabev Hristo , Nixon Marianne , Eastwood Ian M. , Greenhalgh Malcolm , Handley Pauline S. , Robson Geoffrey D.
Published: July 2002

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish
Aequoria victoria
was tested as a potential reporter molecule for this purpose.
Aureobasidium pullulans
was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1
gfp
showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1
gfp
blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (
r
2
> 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-
n
-octylisothiozolin-3-one (OIT) to suspensions of Ap1
gfp
blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml
−1
and 500 μg ml
−1
, respectively. Further, loss of GFP fluorescence from
A. pullulans
cells was highly correlated with a decrease in the number of viable cells (
r
2
> 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When
A. pullulans
was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of
A. pullulans
and other fungal species to antimicrobial compounds.
Purification, Stability, and Mineralization of 3-Hydroxy-2- Formylbenzothiophene, a Metabolite of Dibenzothiophene
Authors: Bressler David C. , Fedorak Phillip M.
Published: July 2002

3-Hydroxy-2-formylbenzothiophene (HFBT) is a metabolite found in many bacterial cultures that degrade dibenzothiophene (DBT) via the Kodama pathway. The fate of HFBT in cultures and in the environment is unknown. In this study, HFBT was produced by a DBT-degrading bacterium and purified by sublimation. When stored in organic solvent or as a crystal, the HFBT slowly decomposed, yielding colored products. Two of these were identified as thioindigo and
cis
-thioindigo. The supernatant of the DBT-degrading culture contained thioindigo, which has not been reported previously as a product of DBT biodegradation. In mineral salts medium, HFBT was sufficiently stable to allow biodegradation studies with a mixed microbial culture over a 3- to 4-week period. High-performance liquid chromatography analyses showed that HFBT was removed from the medium. 2-Mercaptophenylglyoxalate, detected as benzothiophene-2,3-dione, was found in an HFBT-degrading mixed culture, and the former appears to be a metabolite of HFBT. This mixed culture also mineralized HFBT to CO
2
.
Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker
Authors: O' Sullivan David , Ross R. Paul , Twomey Denis P. , Fitzgerald Gerald F. , Hill Colin , Coffey Aidan
Published: July 2002

The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (
hsdS
) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866–876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the
cadA
gene, which has an associated regulatory gene (
cadC
), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.
Biogeochemical and Molecular Signatures of Anaerobic Methane Oxidation in a Marine Sediment
Authors: Thomsen Trine R. , Finster Kai , Ramsing Niels B.
Published: July 2002

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for
Archaea
. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802–805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera
Desulfonema
,
Desulfococcus
, and
Desulfosarcina
. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.
Diversity and Seasonal Fluctuations of the Dominant Members of the Bacterial Soil Community in a Wheat Field as Determined by Cultivation and Molecular Methods
Authors: Smit Eric , Leeflang Paula , Gommans Suzanne , van den Broek Jan , van Mil Saskia , Wernars Karel
Published: July 2002

There is a paucity of knowledge on microbial community diversity and naturally occurring seasonal variations in agricultural soil. For this purpose the soil microbial community of a wheat field on an experimental farm in The Netherlands was studied by using both cultivation-based and molecule-based methods. Samples were taken in the different seasons over a 1-year period. Fatty acid-based typing of bacterial isolates obtained via plating revealed a diverse community of mainly gram-positive bacteria, and only a few isolates appeared to belong to the
Proteobacteria
and green sulfur bacteria. Some genera, such as
Micrococcus
,
Arthrobacter
, and
Corynebacterium
were detected throughout the year, while
Bacillus
was found only in July. Isolate diversity was lowest in July, and the most abundant species,
Arthrobacter oxydans
, and members of the genus
Pseudomonas
were found in reduced numbers in July. Analysis by molecular techniques showed that diversity of cloned 16S ribosomal DNA (rDNA) sequences was greater than the diversity among cultured isolates. Moreover, based on analysis of 16S rDNA sequences, there was a more even distribution among five main divisions,
Acidobacterium
,
Proteobacteria
,
Nitrospira
, cyanobacteria, and green sulfur bacteria. No clones were found belonging to the gram-positive bacteria, which dominated the cultured isolates. Seasonal fluctuations were assessed by denaturing gradient gel electrophoresis. Statistical analysis of the banding patterns revealed significant differences between samples taken in different seasons. Cluster analysis of the patterns revealed that the bacterial community in July clearly differed from those in the other months. Although the molecule- and cultivation-based methods allowed the detection of different parts of the bacterial community, results from both methods indicated that the community present in July showed the largest difference from the communities of the other months. Efforts were made to use the sequence data for providing insight into more general ecological relationships. Based on the distribution of 16S rDNA sequences among the bacterial divisions found in this work and in literature, it is suggested that the ratio between the number of
Proteobacteria
and
Acidobacterium
organisms might be indicative of the trophic level of the soil.
Identification of a Small Tetraheme Cytochrome
c
and a Flavocytochrome
c
as Two of the Principal Soluble Cytochromes
c
in
Shewanella oneidensis
Strain MR1
Authors: Tsapin A. I. , Vandenberghe I. , Nealson K. H. , Scott J. H. , Meyer T. E. , Cusanovich M. A. , Harada E. , Kaizu T. , Akutsu H. , Leys D. , Van Beeumen J. J.
Published: July 2002

Two abundant, low-redox-potential cytochromes
c
were purified from the facultative anaerobe
Shewanella oneidensis
strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome
c
contains 91 residues and four heme binding sites. It is most similar to the cytochromes
c
from
Shewanella frigidimarina
(formerly
Shewanella putrefaciens
) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome
c
-fumarate reductase previously characterized from
S. frigidimarina
to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two
Shewanella
species, and they are not located in multicistronic operons. The small cytochrome
c
and the cytochrome domain of the flavocytochrome
c
are also homologous, showing 34% identity. Structural comparison shows that the
Shewanella
tetraheme cytochromes are not related to the
Desulfovibrio
cytochromes
c
3
but define a new folding motif for small multiheme cytochromes
c
.
Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor
Authors: Gonzalez-Gil G. , Lens P. N. L. , Van Aelst A. , Van As H. , Versprille A. I. , Lettinga G.
Published: July 2002

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH
4
/g of volatile suspended solids [VSS]·day or 1.1 g of CH
4
chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (
Methanosaeta
spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (
Methanobacterium
-like and
Methanospirillum
-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.
Survival of
Lactobacillus delbrueckii
subsp.
bulgaricus
and
Streptococcus thermophilus
in the Terminal Ileum of Fistulated Göttingen Minipigs
Authors: Lick Sonja , Drescher Karsten , Heller Knut J.
Published: July 2002

The ability of
Lactobacillus delbrueckii
subsp.
bulgaricus
and
Streptococcus thermophilus
administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living
L. delbrueckii
subsp.
bulgaricus
and
S. thermophilus
were detected in the magnitude of 10
6
to 10
7
per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for
L. delbrueckii
and
S. thermophilus
and subspecies specific for
L. delbrueckii
subsp.
bulgaricus
. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous
L. delbrueckii
flora. When heat-treated yogurt was administered,
L. delbrueckii
was detected in all animals.
S. thermophilus
or
L. delbrueckii
subsp.
bulgaricus
was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own
L. delbrueckii
flora had been stimulated for growth.
Contribution of
Lactococcus lactis
Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese
Authors: Broadbent Jeffery R. , Barnes Mary , Brennand Charlotte , Strickland Marie , Houck Kristen , Johnson Mark E. , Steele James L.
Published: July 2002

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the
Lactococcus lactis
extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of
Lactococcus lactis
as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of α
S1
-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.
Abnormal CD40 Ligand (CD154) Expression in Human Immunodeficiency Virus-Infected Children
Authors: O'Gorman Maurice R. G. , DuChateau Brian , Paniagua Mary , Hunt Janelle , Bensen Nicolas , Yogev Ram
Published: July 2002

The CD40 ligand (CD154), expressed primarily on activated CD4-positive T cells, is a costimulatory molecule involved in B-cell proliferation, germinal center formation, and immunoglobulin class switching. Since B-cell abnormalities including hypergammaglobulinemia and abnormal antibody-specific immune responses are prominent and occur early during the course of pediatric human immunodeficiency virus (HIV) infection, we measured the baseline levels and the induced levels of expression of CD154 on CD3+CD8−(T helper cells) in HIV-infected children and uninfected children born to HIV-positive mothers. The percentage of CD154+T helper cells activated in vitro and the level of CD154 expressed per T helper cell (mean fluorescent channel [MFC]) were significantly lower in the HIV-infected children than in the uninfected control group (77% ± 3% versus 89% ± 1%, respectively [P< 0.002], and 261 ± 174 versus 415 ± 130 MFC, respectively [P< 0.03]). The levels of CD154 expressed on resting T helper cells in the HIV-infected group were not significantly different from the levels observed in the control group. In the HIV-infected children, the level of CD154 on activated T helper cells correlated with the level of immunodeficiency, as assessed by the CD4 T-cell levels (correlation coefficient [r] = 0.707,P= 0.003), but did not correlate with the viral load or with any of the serum immunoglobulin concentrations measured in this group of HIV-infected children. The baseline level of CD154 expressed on T helper cells did, however, correlate with the concentration of immunoglobulin A in serum. We conclude that HIV-infected children have impaired regulation of CD154 expression which may contribute to the immune dysregulation commonly observed.
Receiver Operating Characteristics Analyses of Food and Drug Administration-Cleared Serological Assays for Natural Rubber Latex-Specific Immunoglobulin E Antibody
Authors: Biagini Raymond E. , Krieg Edward F. , Pinkerton Lynne E. , Hamilton Robert G.
Published: July 2002

Receiver operating characteristics (ROC) analyses to evaluate and compare the diagnostic accuracy of Food and Drug Administration (510K)-cleared natural rubber latex (NRL)-specific immunoglobulin E (IgE) antibody immunoassays have not been performed using well-characterized skin-testing reagents. Sera were collected from 311 subjects (131 latex puncture skin test [PST] positive and 180 PST negative). All masked, coded sera were analyzed for latex-specific IgE antibodies in the Diagnostic Products Corporation microplate AlaSTAT, HYCOR HY-TEC RAST, and Pharmacia-Upjohn CAP System RAST FEIA (CAP). Diagnostic accuracy was evaluated using GraphRoc for Windows software to construct and analyze ROC curves in relation to the subjects' PST status and the results of the immunoassays. The ROC areas under the curve (AUCs) ± standard error based on PST for the three diagnostic tests were 0.858 ± 0.024, 0.869 ± 0.024, and 0.924 ± 0.017, respectively, for AlaSTAT, CAP, and HY-TEC. The HY-TEC system had a significantly greater AUC based on PST than those observed for AlaSTAT (
P
< 0.05) and CAP (
P
< 0.05) analyses. When the diagnostic tests were probed as to the cutoffs giving maximal diagnostic efficiency compared to PST, CAP and AlaSTAT yielded values of <0.35 kU of allergen IgE (kU
A
)/liter and <0.35 kU/liter while the HY-TEC assay yielded 0.11 kU/liter. The diagnostic efficiencies based on PST in our cohort at these cutoffs were 87.1, 88.1, and 88.7%, respectively. The HY-TEC assay had a significantly greater AUC than CAP and AlaSTAT using PST as a diagnostic discriminator in our cohort. When the HY-TEC system was probed at its maximally efficient cutoff (0.11 kU/liter) versus HYCOR's recommended cutoff of 0.05 kU/liter, a loss of sensitivity of 8.4% was observed with a gain in specificity of 19.5%.
Infection with the HelminthNippostrongylus brasiliensisDoes Not Interfere with Efficient Elimination ofMycobacterium bovisBCG from the Lungs of Mice
Authors: Erb Klaus J. , Trujillo Claudia , Fugate Mike , Moll Heidrun
Published: July 2002

Infection withMycobacterium tuberculosiscontinues to be one of the major global health threats. Strong mycobacterium-specific Th1 immune responses correlate with protection, and decreased Th1 responses correlate with disease progression. In contrast, the impact of Th2 responses on the development of protective immune responses to mycobacteria remains unclear. To analyze whether ongoing Th2 responses present in the lung influence the development of a protective Th1 immune response to mycobacteria, we coinfected mice with the helminthNippostrongylus brasiliensisandMycobacterium bovisBCG. We found that the T cells from the lymph nodes of coinfected mice secreted significantly less gamma interferon than did the T cells from mice infected withM. bovisBCG after in vitro stimulation with purified protein fromM. tuberculosiswhen 108CFU ofM. bovisBCG were used for the infection. This result indicates that the helminth infection reduced the Th1 immune response to the mycobacteria in the lung. However, mycobacterial clearance was not delayed in the coinfected animals. Importantly, the infection with BCG after the helminth infection did not reduce the helminth-induced Th2 response in the lung, ruling out the possibility that the lack of a reduction in bacterial clearance in the coinfected mice was due to a downmodulation of the helminth-induced Th2 response. Taken together, our results suggest that ongoing Th2 responses in the lung do not necessarily lead to increased susceptibility to mycobacterial infection.
Sex-Associated Hormones and Immunity to Protozoan Parasites
Authors: Roberts Craig W. , Walker William , Alexander James
Published: July 2002

Numerous epidemiological and clinical studies have noted differences in the incidence and severity of parasitic diseases between males and females. Although in some instances this may be due to gender-associated differences in behavior, there is overwhelming evidence that sex-associated hormones can also modulate immune responses and consequently directly influence the outcome of parasitic infection. Animal models of disease can often recreate the gender-dependent differences observed in humans, and the role of sex-associated hormones can be confirmed by experimentally altering their levels. Under normal circumstances, levels of sex hormones not only differ between males and females but vary according to age. Furthermore, not only are females of reproductive age subject to the regular hormonal cycles which control ovulation, they are also exposed to dramatically altered levels during pregnancy. It is thus not surprising that the severity of many diseases, including those caused by parasites, has been shown to be affected by one or more of these circumstances. In addition, infection with many pathogens has been shown to have an adverse influence on pregnancy. In this article we review the impact of sex-associated hormones on the immune system and the development and maintenance of immunity to the intracellular protozoan parasites Toxoplasma gondii, Plasmodium spp., and Leishmania spp.
Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions
Authors: Chensue Stephen W.
Published: July 2002

Chemokines and their G-protein-coupled receptors represent an ancient and complex system of cellular communication participating in growth, development, homeostasis and immunity. Chemokine production has been detected in virtually every microbial infection examined; however, the precise role of chemokines is still far from clear. In most cases they appear to promote host resistance by mobilizing leukocytes and activating immune functions that kill, expel, or sequester pathogens. In other cases, the chemokine system has been pirated by pathogens, especially protozoa and viruses, which have exploited host chemokine receptors as modes of cellular invasion or developed chemokine mimics and binding proteins that act as antagonists or inappropriate agonists. Understanding microbial mechanisms of chemokine evasion will potentially lead to novel antimicrobial and anti-inflammatory therapeutic agents.
Rapid Diagnostic Tests for Malaria Parasites
Authors: Moody Anthony
Published: July 2002

Malaria presents a diagnostic challenge to laboratories in most countries. Endemic malaria, population movements, and travelers all contribute to presenting the laboratory with diagnostic problems for which it may have little expertise available. Drug resistance and genetic variation has altered many accepted morphological appearances of malaria species, and new technology has given an opportunity to review available procedures. Concurrently the World Health Organization has opened a dialogue with scientists, clinicians, and manufacturers on the realistic possibilities for developing accurate, sensitive, and cost-effective rapid diagnostic tests for malaria, capable of detecting 100 parasites/μl from all species and with a semiquantitative measurement for monitoring successful drug treatment. New technology has to be compared with an accepted Rgold standardS that makes comparisons of sensitivity and specificity between different methods. The majority of malaria is found in countries where cost-effectiveness is an important factor and ease of performance and training is a major consideration. Most new technology for malaria diagnosis incorporates immunochromatographic capture procedures, with conjugated monoclonal antibodies providing the indicator of infection. Preferred targeted antigens are those which are abundant in all asexual and sexual stages of the parasite and are currently centered on detection of HRP-2 from Plasmodium falciparum and parasite-specific lactate dehydrogenase or Plasmodium aldolase from the parasite glycolytic pathway found in all species. Clinical studies allow effective comparisons between different formats, and the reality of nonmicroscopic diagnoses of malaria is considered.
Effects of Interleukin-4 Deprivation and Treatment on Resistance toTrypanosoma cruzi
Authors: Abrahamsohn Ises A. , da Silva Ana Paula Galvão , Coffman Robert L.
Published: July 2002

Trypanosoma cruzi(Y strain)-infected interleukin-4−/−(IL-4−/−) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-γ) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4−/−mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-γ synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-γ synthesis levels; in contrast, IL-10 neutralization increased IFN-γ production and addition of rIL-4 and/or rIL-10 diminished IFN-γ synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strainT. cruziinfection but that IL-4 can, in association with IL-10, modulate IFN-γ production and resistance.
Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of
Porphyromonas gingivalis
Authors: Lopatin Dennis E. , Combs Allison , Sweier Domenica G. , Fenno J. Christopher , Dhamija Sangeeta
Published: July 2002

Porphyromonas gingivalis
is implicated in the etiology of periodontal disease. Associations between microbial virulence and stress protein expression have been identified in other infections. For example, Hsp90 homologues in several microbial species have been shown to contribute to virulence. We previously reported that
P. gingivalis
possessed an Hsp90 homologue (HtpG) which cross-reacts with human Hsp90. In addition, we found that elevated levels of serum antibody to Hsp90 stress protein in individuals colonized with this microorganism were associated with periodontal health. However, the role of HtpG in
P. gingivalis
has not been explored. Therefore, we cloned the
htpG
gene and investigated the characteristics of HtpG localization and expression in
P. gingivalis. htpG
exists as a single gene of 2,052 bp from which a single message encoding a mature protein of approximately 68 kDa is transcribed. Western blot analysis revealed that the 68-kDa polypeptide was stress inducible and that a major band at 44 kDa and a minor band at 40 kDa were present at constitutive levels. Cellular localization studies revealed that the 44- and 40-kDa species were associated with membrane and vesicle fractions, while the 68-kDa polypeptide was localized to the cytosolic fractions.
Nitric Oxide Participation in the Fungicidal Mechanism of Gamma Interferon-Activated Murine Macrophages againstParacoccidioides brasiliensisConidia
Authors: Gonzalez Angel , de Gregori Waldemar , Velez Diana , Restrepo Angela , Cano Luz E.
Published: July 2002

Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungusParacoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form. The macrophage (Mφ) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-γ), murine Mφs kill the fungus by an oxygen-independent mechanism. Our goal was to determine the role of nitric oxide in the fungicidal effect of Mφs onP. brasiliensisconidia. The results revealed that IFN-γ-activated murine Mφs inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mφs activated with 50 U/ml and incubated for 96 h at 37°C. When Mφs were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect. The inhibitory effect was reversed by the addition of anti-IFN-γ monoclonal antibodies. Activation by IFN-γ also enhanced Mφ nitric oxide production, as revealed by increasing NO2values (8 ± 3 μM in nonactivated Mφs versus 43 ± 13 μM in activated Mφs). The neutralization of IFN-γ also reversed nitric oxide production at basal levels (8 ± 5 μM). Additionally, we found that there was a significant inverse correlation (r= −0.8975) between NO2−concentration and transformation ofP. brasiliensisconidia. Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase,NG-monomethyl-l-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production. These results show that IFN-γ-activated murine Mφs killP. brasiliensisconidia through thel-arginine–nitric oxide pathway.
Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis
Authors: Schaefer Deborah A. , Auerbach-Dixon Beth A. , Riggs Michael W.
Published: July 2002

The coccidian parasiteCryptosporidium parvumcauses diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against wholeC. parvumreduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.
Classical and El Tor Biotypes of
Vibrio cholerae
Differ in Timing of Transcription of
tcpPH
during Growth in Inducing Conditions
Authors: Murley Yvette M. , Behari Jaideep , Griffin Robert , Calderwood Stephen B.
Published: July 2002

Two protein pairs in
Vibrio cholerae
, ToxRS and TcpPH, are necessary for transcription from the
toxT
promoter and subsequent expression of cholera virulence genes. We have previously shown that transcription of
tcpPH
in classical strains of
V. cholerae
is activated at mid-log-phase growth in ToxR-inducing conditions, while transcription of
tcpPH
in El Tor strains is not. In this study, we showed that while transcription of
tcpPH
differs at mid-log-phase growth in ToxR-inducing conditions between the biotypes, transcription is equivalently high during growth in AKI conditions. We used
tcpPH
::
gusA
transcriptional fusions to quantitate expression of
tcpPH
in each biotype throughout growth in ToxR-inducing conditions and showed that although transcription of
tcpPH
is reduced at mid-log-phase growth in an El Tor strain, transcription is turned on later in growth to levels in excess of those in the classical strain (although cholera toxin is not produced). This suggests that the difference in expression of cholera virulence factors in response to ToxR-inducing conditions between the El Tor and classical biotypes of
V. cholerae
may be related to the timing of transcription of
tcpPH
rather than the absolute levels of transcription.
Inhibitory and Bactericidal Effects of Hydrogen Peroxide Production byStreptococcus pneumoniaeon Other Inhabitants of the Upper Respiratory Tract
Authors: Pericone Christopher D. , Overweg Karin , Hermans Peter W. M. , Weiser Jeffrey N.
Published: July 2002

An inverse correlation between colonization of the human nasopharynx byStreptococcus pneumoniaeandHaemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants fromS. pneumoniaeinhibited growth ofH. influenzae, whereas culture supernatants fromH. influenzaehad no effect on the growth ofS. pneumoniae. Moreover, coculture ofS. pneumoniaeandH. influenzaeled to a rapid decrease in viable counts ofH. influenzae. The addition of purified catalase prevented killing ofH. influenzaein coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity.H. influenzaewas killed by concentrations of hydrogen peroxide similar to that produced byS. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both anspxBmutant and a naturally occurring variant ofS. pneumoniae, which is downregulated in SpxB expression, were unable to killH. influenzae. A catalase-reversible inhibitory effect ofS. pneumoniaeon the growth of the respiratory tract pathogensMoraxella catarrhalisandNeisseria meningitidiswas also observed. Elevated hydrogen peroxide production, therefore, may be a means by whichS. pneumoniaeis able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.
Aromatic Compound-Dependent
Brucella suis
Is Attenuated in Both Cultured Cells and Mouse Models
Authors: Foulongne Vincent , Walravens Karl , Bourg Giséle , Boschiroli Maria Laura , Godfroid Jacques , Ramuz Michel , O'Callaghan David
Published: July 2002

The
aroC
gene of the facultative intracellular pathogen
Brucella suis
was cloned and sequenced. The cloned
aroC
gene complements
Escherichia coli
and
Salmonella enterica
serovar Typhimurium
aroC
mutants. A
B. suis aroC
mutant was found to be unable to grow in a defined medium without aromatic compounds. The mutant was highly attenuated in tissue culture (THP1 macrophages and HeLa cells) and murine virulence models.
Differential Expression of the
Aspergillus fumigatus pksP
Gene Detected In Vitro and In Vivo with Green Fluorescent Protein
Authors: Langfelder Kim , Philippe Bruno , Jahn Bernhard , Latgé Jean-Paul , Brakhage Axel A.
Published: July 2002

Aspergillus fumigatus
is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of
A. fumigatus
, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5′ sequences, containing the putative promoters of the
pyrG
gene, encoding orotidine-5′-phosphate decarboxylase, and the
pksP
gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence of
A. fumigatus
, were fused with the
egfp
gene. The P
pksP
-
egfp
construct was integrated via homologous recombination into the genomic
pksP
locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in
A. fumigatus
was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly,
pksP
-
egfp
expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that the
pksP
gene can also be expressed in hyphae under certain conditions and, furthermore, that the
pksP
gene might also contribute to invasive growth of the fungus.
Identification of New Genes Involved in the Virulence ofListeria monocytogenesby Signature-Tagged Transposon Mutagenesis
Authors: Autret Nicolas , Dubail Iharilalao , Trieu-Cuot Patrick , Berche Patrick , Charbit Alain
Published: July 2002

Listeria monocytogenesis a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis toL. monocytogenesto identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks ofL. monocytogenesmutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.
Protection againstPseudomonas aeruginosaChronic Lung Infection in Mice by Genetic Immunization against Outer Membrane Protein F (OprF) ofP. aeruginosa
Authors: Price Brian M. , Galloway Darrell R. , Baker Neil R. , Gilleland Linda B. , Staczek John , Gilleland Harry E.
Published: July 2002

ThePseudomonas aeruginosamajor constitutive outer membrane porin protein OprF, which has previously been shown to be a protective antigen, was targeted as a DNA vaccine candidate. TheoprFgene was cloned into plasmid vector pVR1020, and the plasmid vaccines were delivered to mice by biolistic (gene gun) intradermal inoculation. Antibody titers in antisera from immunized mice were determined by enzyme-linked immunosorbent assay, and the elicited antibodies were shown to be specifically reactive to OprF by immunoblotting. The immunoglobulin G (IgG) immune response was predominantly of the IgG1 isotype. Sera from DNA vaccine-immunized mice had significantly greater opsonic activity in opsonophagocytic assays than did sera from control mice. Following the initial immunization and two consecutive boosts, each at 2-week intervals, protection was demonstrated in a mouse model of chronic pulmonary infection byP. aeruginosa. Eight days postchallenge, both lungs were removed and examined. A significant reduction in the presence of severe macroscopic lesions, as well as in the number of bacteria present in the lungs, was seen. Based on these findings, genetic immunization withoprFhas potential for development as a vaccine to protect humans against infection byP. aeruginosa.
Effects of Exogenous Interleukin-6 during
Pseudomonas aeruginosa
Corneal Infection
Authors: Cole Nerida , Krockenberger Mark , Bao Shisan , Beagley Kenneth W. , Husband Alan J. , Willcox Mark
Published: July 2002

Lack of interleukin-6 (IL-6) during
Pseudomonas aeruginosa
corneal infection leads to more severe disease with changes in neutrophil recruitment. Exogenous IL-6 leads to increased efficiency of neutrophil recruitment and reduced bacterial loads in corneal infection in both IL-6 gene knockout and wild-type mice. This may be mediated by IL-6 increasing the production of corneal macrophage inflammatory protein 2 and intercellular cell adhesion molecule 1. We conclude that effective recruitment of neutrophils into the cornea is dependent on the production of IL-6 and that early augmentation of IL-6 may be protective in corneal infection.
Protection of BALB/c Mice againstBrucella abortus544 Challenge by Vaccination with Bacterioferritin or P39 Recombinant Proteins with CpG Oligodeoxynucleotides as Adjuvant
Authors: Al-Mariri Ayman , Tibor Anne , Mertens Pascal , De Bolle Xavier , Michel Patrick , Godefroid Jacques , Walravens Karl , Letesson Jean-Jacques
Published: July 2002

The P39 and the bacterioferrin (BFR) antigens ofBrucella melitensis16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-γ) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine againstBrucella abortusinfection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-γ production and T-cell proliferation in response to eitherBrucellaextract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using aB. abortus544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.
Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis
Authors: Wormley Floyd L. , Chaiban Joseph , Fidel Paul L.
Published: July 2002

Cell-mediated immunity by Th1-type CD4+T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence ofCandida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondaryC. albicansvaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginalCandidainfection, CD3+cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginalCandidainfection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limitingCandida-specific T-cell responses against infection.