bluuebunny/crossref_metadata_2025_split huggingface dataset:
An OmpA-Like Protein fromAcinetobacterspp. Stimulates Gastrin and Interleukin-8 Promoters
Authors: Ofori-Darko Ernest , Zavros Yana , Rieder Gabriele , Tarlé Susan A. , Van Antwerp Mary , Merchant Juanita L. 
Published: July 2002

ABSTRACTBacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 108organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified anAcinetobactersp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of theAcinetobactersp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.
Regulation of
Brucella abortus
Catalase
Authors: Kim Jeong-a , Sha Zengyu , Mayfield John E. 
Published: July 2002

ABSTRACT

All aerobic organisms have mechanisms that protect against oxidative compounds. Catalase, peroxidase, superoxide dismutase, glutathione, and thioredoxin are widely distributed in many taxa and constitute elements of a nearly ubiquitous antioxidant metabolic strategy. Interestingly, the regulatory mechanisms that control these elements are rather different depending on the nature of the oxidative stress and the organism. Catalase is well documented to play an important role in protecting cells from oxidative stress. In particular, pathogenic bacteria seem to use this enzyme as a defensive tool against attack by the host. To investigate the significance of catalase in hostile environments, we made catalase deletion mutations in two different
B. abortus
strains and used two-dimensional gel analysis, survival tests, and adaptation experiments to explore the behavior and role of catalase under several oxidative stress conditions. These studies show that
B. abortus
strains that do not express catalase activity exhibit increased sensitivity to hydrogen peroxide. We also demonstrate that catalase expression is regulated in this species, and that preexposure to a sublethal concentration of hydrogen peroxide allows
B. abortus
to adapt so as to survive subsequent exposure to higher concentrations of hydrogen peroxide.
Secreted Aspartic Proteinase Family of
Candida tropicalis
Authors: Zaugg Christophe , Borg-von Zepelin Margarete , Reichard Utz , Sanglard Dominique , Monod Michel 
Published: July 2002

ABSTRACT

Medically important yeasts of the genus
Candida
secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like
Candida albicans
,
Candida tropicalis
secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene
SAPT1
as a probe and under low-stringency hybridization conditions, three new closely related gene sequences,
SAPT2
to
SAPT4
, encoding secreted proteinases were cloned from a
C. tropicalis
λEMBL3 genomic library. All bands identified by Southern blotting of
Eco
RI-digested
C. tropicalis
genomic DNA with
SAPT1
could be assigned to a specific
SAP
gene. Therefore, the
SAPT
gene family of
C. tropicalis
is likely to contain only four members. Interestingly, the
SAPT2
and
SAPT3
gene products, Sapt2p and Sapt3p, which have not yet been detected in
C. tropicalis
cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast
Pichia pastoris
as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong
SAPT1
signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other
SAPT
genes under several conditions tested, showing that these
SAPT
genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.
Ferrochelatase Is Present in
Brucella
abortus
and Is Critical for Its Intracellular Survival and Virulence
Authors: Almirón Marta , Martı́nez Marcela , Sanjuan Norberto , Ugalde Rodolfo A. 
Published: July 2002

ABSTRACT

Brucella
spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established,
Brucella
spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of
Brucella
abortus
. During a
B
.
abortus
genomic sequencing project, a clone coding a putative gene homologous to
hemH
was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type
hemH
. These data demonstrate that
B
.
abortus
synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of
B
.
abortus
and thus for the establishment of chronic disease as well.
Regulation of the Cytotoxic Enterotoxin Gene in
Aeromonas hydrophila
: Characterization of an Iron Uptake Regulator
Authors: Sha Jian , Lu Muping , Chopra Ashok K. 
Published: July 2002

ABSTRACT

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of
Aeromonas hydrophila
is aerolysin related and crucial to the pathogenesis of
Aeromonas
infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (
act
), a portion of the
act
gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (
phoA
) of
Escherichia coli
. The
act
::
phoA
reporter gene was then introduced into the chromosome of
A. hydrophila
by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the
act
promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The
act
promoter exhibited optimal activity at pH 7.0 and at 37°C, and maximal PhoA activity was noted when the culture was aerated. Using a
Vibrio cholerae
iron uptake regulator gene (
fur
) as a probe, a 2.6-kb
Sal
I/
Hin
dIII DNA fragment from an
A. hydrophila
chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the
fur
gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of
V. cholerae
. Complementation experiments demonstrated that the
A. hydrophila fur
gene could restore iron regulation in an
E. coli fur
-minus mutant. Using the suicide vector pDMS197, we generated a
fur
isogenic mutant of wild-type
A. hydrophila
SSU. Northern blot analysis data indicated that the repression in the transcription of the
act
gene by iron was relieved in the
fur
isogenic mutant. Further, iron regulation in the
fur
isogenic mutant of
A. hydrophila
could be restored by complementation. These results are important in understanding the regulation of the
act
gene under in vivo conditions.
Potential Role for Extracellular Glutathione-Dependent Ferric Reductase in Utilization of Environmental and Host Ferric Compounds byHistoplasma capsulatum
Authors: Timmerman Michelle M. , Woods Jon P. 
Published: July 2002

ABSTRACTThe mammalian host specifically limits iron duringHistoplasma capsulatuminfection, and fungal acquisition of iron is essential for productive infection.H. capsulatumexpresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. TheH. capsulatumsiderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).
VraA (BBI16) Protein of
Borrelia burgdorferi
Is a Surface-Exposed Antigen with a Repetitive Motif That Confers Partial Protection against Experimental Lyme Borreliosis
Authors: Labandeira-Rey Maria , Baker Elizabeth A. , Skare Jonathan T. 
Published: July 2002

ABSTRACT

We have previously described the expression cloning of nine
Borrelia burgdorferi
antigens, using rabbit serum enriched for antibodies specific for infection-associated antigens, and determined that seven of these antigens were associated with infectious
B. burgdorferi
strain B31. One of these infection-associated antigens encoded a 451-amino-acid putative lipoprotein containing 21 consecutive and invariant 9-amino-acid repeat sequences near the amino terminus that we have designated VraA for virulent strain-associated repetitive antigen A. The
vraA
locus (designated BBI16 by The Institute for Genomic Research) maps to one of the 28-kb linear plasmids (designated lp28-4) that is not present in noninfectious strain B31 isolates. Subsequent PCR analysis of clonal isolates of
B. burgdorferi
B31 from infected mouse skin revealed a clone that lacked only lp28-4. Southern blot and Western blot analyses indicated that the lp28-4 and VraA proteins, respectively, were missing from this clone. We have also determined that VraA is a surface-exposed protein based on protease accessibility assays of intact whole cells. Furthermore,
vraA
expression is modestly derepressed when cells are grown at 37°C relative to cells grown at 32°C, suggesting that VraA is, in part, a temperature-inducible antigen. Homologues cross-reactive to
B. burgdorferi
B31 VraA, most with different molecular masses, were identified in several
B. burgdorferi
sensu lato isolates, including
B. andersonii
, suggesting that the immunogenic epitope(s) present in strain B31 VraA is conserved between
Borrelia
spp. In protection studies, only 8.3% of mice (1 of 12) immunized with full-length recombinant VraA fused to glutathione
S
-transferase (GST) were susceptible to infectious challenge with 10
2
B. burgdorferi
strain B31, whereas naive mice or mice immunized with GST alone were infected 40% or 63 to 67% (depending on tissues assayed) of the time, respectively. As such, the partial protection elicited by VraA immunization provides an additional testable vaccine candidate to help protect against Lyme borreliosis.
cDNA Array Analysis of Altered Gene Expression in Human Endothelial Cells in Response toChlamydia pneumoniaeInfection
Authors: Coombes Brian K. , Mahony James B. 
Published: July 2002

ABSTRACTStrong epidemiological and pathological evidence supports a role forChlamydia pneumoniaeinfection in atherosclerosis and human coronary heart disease. Animal models have shown thatC. pneumoniaedisseminates hematogenously in infected monocytes and macrophages, while in vitro data suggest that infected macrophages can transmitC. pneumoniaeinfection directly to endothelial cells. Endothelial cells may be key in vivo targets forC. pneumoniaeinfection; given that these cells are important in regulating the dynamics of the vessel wall, we used cDNA microarrays to study the transcriptional response of endothelial cells to infection withC. pneumoniae. cDNA arrays were used to characterize the mRNA expression profiles for 268 human genes following infection withC. pneumoniae, which were compared to mRNA profiles of uninfected cells. Selected genes of interest were further investigated by reverse transcription-PCR throughout a 24-h period of infection.C. pneumoniaeinfection upregulated mRNA expression for approximately 20 (8%) of the genes studied. Genes coding for cytokines (interleukin-1), chemokines (monocyte chemotactic protein 1 and interleukin-8), and cellular growth factors (heparin-binding epidermal-like growth factor, basic fibroblast growth factor, and platelet-derived growth factor B chain) were the most prominently upregulated. In addition to these families of genes, increases in mRNA levels for intracellular kinases and cell surface receptors with signal transduction activities were observed. Time course experiments showed that mRNA levels were upregulated within 2 h following infection. These results expand our knowledge of the response of endothelial cells toC. pneumoniaeby further defining the repertoire ofC. pneumoniae-inducible genes and provide new insight into potential mechanisms of atherogenesis. In addition, the use of cDNA microarrays may prove useful for the study of host cell responses toC. pneumoniaeinfection during latent and replicative stages of infection and related pathology.
Cellular Basis of Early Cytokine Response toPlasmodium falciparum
Authors: Hensmann Meike , Kwiatkowski Dominic 
Published: July 2002

ABSTRACTUncertainty remains about the cellular origins of the earliest phase of the proinflammatory cytokine response to malaria. Here we show by fluorescence-activated cell sorter analysis that γδ T cells and CD14+cells from nonimmune donors produce tumor necrosis factor and that γδ T cells also produce gamma interferon within 18 h of contact with mycoplasma-freePlasmodium falciparum-infected erythrocytes in vitro. This early cytokine response is more effectively induced by intact than by lysed parasitized erythrocytes. However, the IFN-γ response to lysed parasites is considerably enhanced several days after peripheral blood mononuclear cells are primed with low numbers of intact parasitized erythrocytes, and in this case it derives from both αβ and γδ T cells. These data show that naı̈ve γδ T cells can respond very rapidly to malaria infection but that malaria fever may involve a multistage process in which the priming of both γδ and αβ T-cell populations boosts the cytokine response to lysed parasite products released at schizont rupture.
SarS, a SarA Homolog Repressible by
agr
, Is an Activator of Protein A Synthesis in
Staphylococcus aureus
Authors: Cheung Ambrose L. , Schmidt Katherine , Bateman Brian , Manna Adhar C. 
Published: July 2002

ABSTRACT

The expression of protein A (
spa
) is repressed by global regulatory loci
sarA
and
agr
. Although SarA may directly bind to the
spa
promoter to downregulate
spa
expression, the mechanism by which
agr
represses
spa
expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the
spa
gene. The expression of
sarS
was almost undetectable in parental strain RN6390 but was highly expressed in
agr
and
sarA
mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a
sarS
mutant as detected in an immunoblot but returned to near-parental levels in a complemented
sarS
mutant. Transcriptional fusion studies with a 158- and a 491-bp
spa
promoter fragment linked to the
xylE
reporter gene disclosed that the transcription of the
spa
promoter was also downregulated in the
sarS
mutant compared with the parental strain. Interestingly, the enhancement in
spa
expression in an
agr
mutant returned to a near-parental level in the
agr sarS
double mutant but not in the
sarA sarS
double mutant. Correlating with this divergent finding is the observation that enhanced
sarS
expression in an
agr
mutant was repressed by the
sarA
locus supplied in
trans
but not in a
sarA
mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp
spa
promoter. Taken together, these data indicated that the
agr
locus probably mediates
spa
repression by suppressing the transcription of
sarS
, an activator of
spa
expression. However, the pathway by which the
sarA
locus downregulates
spa
expression is
sarS
independent.
Activation of Protein Tyrosine Kinases byCoxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis
Authors: Meconi Sonia , Capo Christian , Remacle-Bonnet Maryse , Pommier Gilbert , Raoult Didier , Mege Jean-Louis 
Published: July 2002

ABSTRACTCoxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) byC. burnetiiin THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflectsC. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation inC. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced byC. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, preventedC. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulentC. burnetiibut had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation byC. burnetiinegatively regulates bacterial uptake by interfering with cytoskeleton organization.
Oral Immunization with RecombinantStreptococcus gordoniiExpressingPorphyromonas gingivalisFimA Domains
Authors: Sharma Ashu , Honma Kiyonobu , Evans Richard T. , Hruby Dennis E. , Genco Robert J. 
Published: July 2002

ABSTRACTPorphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis.P. gingivalisfimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation.P. gingivalisfimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development againstP. gingivalis-associated periodontitis. We have previously shown that biologically active domains ofP. gingivalisfimbrillin can be expressed on the surface of the human commensal bacteriumStreptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with twoS. gordoniirecombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes ofP. gingivalisFimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss followingP. gingivalisinfection was also evaluated. The results of this study show that the oral delivery ofP. gingivalisFimA epitopes viaS. gordoniivectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequentP. gingivalis-induced alveolar bone loss. These results support the potential usefulness of theS. gordoniivectors expressingP. gingivalisfimbrillin as a mucosal vaccine against adult periodontitis.
Coinfection withBorrelia burgdorferiand the Agent of Human Granulocytic Ehrlichiosis Alters Murine Immune Responses, Pathogen Burden, and Severity of Lyme Arthritis
Authors: Thomas Venetta , Anguita Juan , Barthold Stephen W. , Fikrig Erol 
Published: July 2002

ABSTRACTLyme disease and human granulocytic ehrlichiosis (HGE) are tick-borne illnesses caused byBorrelia burgdorferiand the agent of HGE, respectively. We investigated the influence of dual infection withB. burgdorferiand the HGE agent on the course of murine Lyme arthritis and granulocytic ehrlichiosis. Coinfection resulted in increased levels of both pathogens and more severe Lyme arthritis compared with those in mice infected withB. burgdorferialone. The increase in bacterial burden during dual infection was associated with enhanced acquisition of both organisms by larval ticks that were allowed to engorge upon infected mice. Coinfection also resulted in diminished interleukin-12 (IL-12), gamma interferon (IFN-γ), and tumor necrosis factor alpha levels and elevated IL-6 levels in murine sera. During dual infection, IFN-γ receptor expression on macrophages was also reduced, implying a decrease in phagocyte activation. These results suggest that coinfection of mice withB. burgdorferiand the HGE agent modulates host immune responses, resulting in increased bacterial burden, Lyme arthritis, and pathogen transmission to the vector.