bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTListeria 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

ABSTRACTThePseudomonas 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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTCell-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.
HLA Class II Polymorphism Influences Onset and Severity of Pathology inSchistosoma mansoni-Infected Transgenic Mice
Authors: Angyalosi Gerhild , Neveu Raphaële , Wolowczuk Isabelle , Delanoye Anne , Herno Josiane , Auriault Claude , Pancré Véronique 
Published: July 2002

ABSTRACTGenetic factors that might influence susceptibility or resistance in naive individuals and early-stage pathology in schistosomiasis are difficult to study in clinical trials, since in areas where the disease is endemic the first contact with the parasite occurs most often at very early ages. Therefore, four strains (DR1.Aβ°, DR2.Aβ°, DQ8.Aβ°, and DQ6.Aβ°) of major histocompatibility complex class II-deficient mice (Aβ°), transgenic for different HLA alleles, have been used to evaluate the potential role of HLA class II polymorphism in the onset of the infection bySchistosoma mansoni. The survival rates and parasitological and immunological parameters after infection were evaluated and compared against the control values obtained with Aβ° mice. All four mouse strains used in this study were able to generate a specific immune response againstS. mansoniantigens (cytokine production and antibody production). However, only mice expressing DR alleles survived until the chronic stage of the infection and were able to mount protective granulomatous response avoiding hepatic damage, presenting predominant gamma interferon production. In contrast, strains expressing DQ alleles revealed an impairment in generating effective granulomas, resulting in earlier death, which was associated with an impaired hepatic granulomatous response and liquefactic necrosis, reflecting the influence of HLA polymorphism in the establishment of protective response in the early stage of infection.
Identification of a Promiscuous T-Cell Epitope inMycobacterium tuberculosisMce Proteins
Authors: Panigada Maddalena , Sturniolo Tiziana , Besozzi Giorgio , Boccieri Maria Giovanna , Sinigaglia Francesco , Grassi Giuliana Gialdroni , Grassi Fabio 
Published: July 2002

ABSTRACTThe characterization ofMycobacterium tuberculosisantigens inducing CD4+T-cell responses could critically contribute to the development of subunit vaccines forM. tuberculosis.Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of themcegenes ofM. tuberculosis.The analysis of the proliferative responses of CD4+T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4+cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.
Fibronectin Attachment Protein Is Necessary for Efficient Attachment and Invasion of Epithelial Cells by
Mycobacterium avium
subsp.
paratuberculosis
Authors: Secott T. E. , Lin T. L. , Wu C. C. 
Published: July 2002

ABSTRACT

Attachment and ingestion of
Mycobacterium avium
subsp.
paratuberculosis
by two epithelial cell lines were enhanced by soluble fibronectin (FN). Peptide blocking of the FN attachment protein (FAP-P) inhibited the internalization of
M. avium
subsp.
paratuberculosis.
Disruption of FAP-P expression significantly reduced attachment and ingestion of
M. avium
subsp.
paratuberculosis
by T-24 and Caco-2 cells. The results indicate that the interaction between FN and FAP-P facilitates attachment and internalization of
M. avium
subsp.
paratuberculosis
by epithelial cells.
Macrophage-Induced Genes ofLegionella pneumophila:Protection from Reactive Intermediates and Solute Imbalance during Intracellular Growth
Authors: Rankin Susannah , Li Zhiru , Isberg Ralph R. 
Published: July 2002

ABSTRACTA promoter-probe strategy was devised to identify genes specifically expressed byLegionella pneumophiladuring growth within the macrophage. Random fragments from theL. pneumophilachromosome were inserted upstream of a promoterless phage T4tdgene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes theL. pneumophilaortholog ofahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis ofahpCgene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate ofL. pneumophilain cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.
Identification of an Alternative Nucleoside Triphosphate: 5′-Deoxyadenosylcobinamide Phosphate Nucleotidyltransferase inMethanobacterium thermoautotrophicumΔH
Authors: Thomas Michael G. , Escalante-Semerena Jorge C. 
Published: July 2002

ABSTRACTComputer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5′-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU inSalmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of theS. enterica cobUgene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domainArchaea. The corresponding ORF open reading frame fromMethanobacterium thermoautotrophicumΔH (MTH1152; referred to ascobY) was amplified and cloned, and the CobY protein was expressed and purified fromEscherichia colias a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme ofS. enterica. NTP:adenosylcobinamide kinase activity was not detected inM. thermoautotrophicumΔH cell extract, suggesting that this organism may not have this activity. ThecobYgene complemented acobUmutant ofS. entericagrown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis.cobY, however, failed to restore adenosylcobalamin biosynthesis incobUmutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.
A Two-Dimensional Protein Gel Electrophoresis Study of the Heat Stress Response of
Bacillus subtilis
Cells during Sporulation
Authors: Movahedi Sara , Waites William 
Published: July 2002

ABSTRACT

The heat resistance of spores of
Bacillus subtilis
formed at 30°C was enhanced by pretreatment at 48°C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.
IS
1675
, a Novel Lactococcal Insertion Element, Forms a Transposon-Like Structure Including the Lacticin 481 Lantibiotic Operon
Authors: Dufour Alain , Rincé Alain , Uguen Patricia , Le Pennec Jean-Paul 
Published: July 2002

ABSTRACT

Two copies of IS
1675
, a novel lactococcal insertion element from the IS
4
family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn
5721
. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS
1675
copies not associated with the lacticin 481 operon. The conservation of IS
1675
flanking sequences suggested a 24-bp target site.
Membrane Topology of the
Bacillus subtilis
Pro-ς
K
Processing Complex
Authors: Green David H. , Cutting Simon M. 
Published: July 2002

ABSTRACT

Activation of the final sporulation-specific transcription factor, ς
K
, is regulated by a signal emanating from the forespore which interacts with the pro-ς
K
processing complex, comprising SpoIVFA, BofA, and the pro-ς
K
processing protease, SpoIVFB. Mature ς
K
then directs late gene expression in the parental compartment of the developing sporangial cell. The nature of this complex and how it is activated to process pro-ς
K
are not understood. All three proteins are predicted to be integral membrane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of
spoIVFA
and
spoIVFB
with the complementary reporters
phoA
and
lacZ
and analyzing activity in
Escherichia coli
. SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in the membrane. SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB. Although the
bofB8
allele, which uncouples the ς
K
checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-ς
K
.
Transport of C
4
-Dicarboxylates in
Wolinella succinogenes
Authors: Ullmann Roland , Gross Roland , Simon Jörg , Unden Gottfried , Kröger Achim 
Published: July 2002

ABSTRACT

C
4
-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in
Wolinella succinogenes
, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane.
W. succinogenes
was found to transport C
4
-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10
3
. The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na
+
, whereas the electrogenic antiport also operated in the absence of Na
+
. In the absence of Na
+
, no electrochemical proton potential (Δp) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (
dcuA
,
dcuB
, and
dctPQM
) encoding putative C
4
-dicarboxylate transporters were identified on the genome of
W. succinogenes
. The predicted gene products of
dcuA
and
dcuB
are similar to the Dcu transporters that are involved in the fumarate respiration of
Escherichia coli
with external C
4
-dicarboxylates. The genes
dctP
, -
Q
, and -
M
probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA
−
DcuB
−
) of
W. succinogenes
lacking the intact
dcuA
and
dcuB
genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA
−
, DcuB
−
, and DctQM
−
mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA
−
DcuB
−
mutant performed fumarate respiration without generating a proton potential even in the presence of Na
+
. This explains why the DcuA
−
DcuB
−
mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na
+
-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.