bluuebunny/crossref_metadata_2025_split huggingface dataset:
BhuR, a Virulence-Associated Outer Membrane Protein of
Bordetella avium
, Is Required for the Acquisition of Iron from Heme and Hemoproteins
Authors: Murphy Erin R. , Sacco Randy E. , Dickenson Amy , Metzger Daniel J. , Hu Yan , Orndorff Paul E. , Connell Terry D. 
Published: September 2002

ABSTRACT

Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium
Bordetella avium
, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen
Bordetella pertussis
. We describe a
B. avium
Fe utilization locus comprised of
bhuR
and six accessory genes (
rhuIR
and
bhuSTUV
). Genetic manipulations of
B. avium
confirmed that
bhuR
, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for
rhuIR
-dependent regulated expression of
bhuR
, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of
B. avium
: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in
hemA
was engineered in
B. avium
to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.
Characterization of a Novel Intracellularly Activated Gene from
Salmonella enterica
Serovar Typhi
Authors: Basso Holger , Rharbaoui Faiza , Staendner Lothar H. , Medina Eva , García-Del Portillo Francisco , Guzmán Carlos A. 
Published: September 2002

ABSTRACT

A
Salmonella enterica
serovar Typhi gene that is selectively up-regulated upon bacterial invasion of eukaryotic cells was characterized. The open reading frame encodes a 298-amino-acid hydrophobic polypeptide (30.8 kDa), which is predicted to be an integral membrane protein with nine membrane-spanning domains. The protein is closely related (87 to 94% reliability) to different transport and permease systems. Gene expression under laboratory conditions was relatively weak; however, sevenfold induction was observed in a high-osmolarity medium (300 mM NaCl). The growth pattern in a laboratory medium of a serovar Typhi strain Ty2 derivative containing a 735-bp in-frame deletion in this gene, named
gaiA
(for gene activated intracellularly), was not affected. In contrast, the mutant was partially impaired in intracellular survival in murine peritoneal macrophages, as well as in human monocyte-derived macrophages. However, in the case of human macrophages, this survival defect was modest and evident only at late infection times (24 h). Despite the distinct intracellular survival kinetics displayed in macrophages of different species, the
gaiA
null mutant was significantly affected in its potential to trigger apoptosis in both murine and human macrophages. Provision of the
gaiA
gene in
trans
resulted in complementation of these phenotypes. Interestingly, the absence of a functional
gaiA
gene caused a marked attenuation in the mouse mucin model, as shown by the increase (3 orders of magnitude) in the 50% lethal dose of the mutant strain over that of the parental strain Ty2 (
P
≤ 0.05). Altogether, these data indicate that the product encoded by the
gaiA
gene is required for triggering apoptosis and bacterial survival within murine macrophages, which is consistent with the in vivo results obtained in the mouse mucin model. However,
gaiA
was not required for initial intracellular survival in human cells, indicating that its role in the natural host might be more complex than is suggested by the studies performed in the murine system.
Selective Accumulation of Mature Asexual Stages of
Plasmodium falciparum
-Infected Erythrocytes in the Placenta
Authors: Beeson James G. , Amin Nishal , Kanjala Maxwell , Rogerson Stephen J. 
Published: September 2002

ABSTRACT

A feature of malaria in pregnancy is accumulation of
P. falciparum
-infected erythrocytes (IEs) in the placenta, which is associated with adverse outcomes for mothers and infants. Infection appears to involve parasite adhesion to molecules such as chondroitin sulfate A, hyaluronic acid, and immunoglobulins. In vitro, adhesion is predominantly a property of mature asexual forms of IEs; however, adhesion of immature or ring forms has recently been reported. We have assessed the parasitemia and developmental stages of IEs in the placenta by examination of placental blood and histological sections with comparison to parasites in the peripheral blood from the same individuals. Approximately 90% of IEs in the placenta were mature forms. Compared to peripheral blood, the placental parasitemia was 10-fold higher and the density of mature IEs was over 200-fold higher. By contrast, the average peripheral and placental ring-stage parasitemias were not significantly different. In 2 of 14 cases, the density of ring forms was higher in placental than in peripheral blood. These findings demonstrate prominent selective accumulation of mature asexual-stage IEs but infrequent accumulation of ring stages in the placental blood spaces, consistent with an important role for mature-stage IE adhesion.
Identification and Characterization of
lpfABCC
′
DE
, a Fimbrial Operon of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Torres Alfredo G. , Giron Jorge A. , Perna Nicole T. , Burland Valerie , Blattner Fred R. , Avelino-Flores Fabiola , Kaper James B. 
Published: September 2002

ABSTRACT

The mechanisms underlying the adherence of
Escherichia coli
O157:H7 and other enterohemorrhagic
E. coli
(EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated
lpfABCC
′
DE
) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (
lpf
) of
Salmonella enterica
serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that
lpfABCC
′
DE
is organized as an operon and that its expression is induced during the exponential growth phase. The
lpf
genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim
−
)
E. coli
K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic
E. coli
O157:H7
lpf
mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the
lpf
operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of
E. coli
O157:H7 with eukaryotic cells by assisting in microcolony formation.
Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by
hemB
Mutants of
Staphylococcus aureus
Expressing Small Colony Variant Phenotypes
Authors: Vaudaux Pierre , Francois Patrice , Bisognano Carmelo , Kelley William L. , Lew Daniel P. , Schrenzel Jacques , Proctor Richard A. , McNamara Peter J. , Peters G. , Von Eiff Christof 
Published: September 2002

ABSTRACT

Small colony variants (SCVs) of
Staphylococcus aureus
are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a
hemB
mutant of
S. aureus
8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of
clfA
and
fnb
genes by the
hemB
mutant compared to its isogenic parent. The influence of the
hemB
mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the
hemB
mutant, either by complementing with intact
hemB
or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the
hemB
mutation occurred independently from
agr
, a major regulatory locus of virulence factors in
S. aureus
. Both
agr
-positive and
agr
-lacking
hemB
mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins.
Fimbrial
ghf
Gene Cluster of Genital Strains of
Haemophilus
spp
Authors: Bruant Guillaume , Gousset Nathalie , Quentin Roland , Rosenau Agnes 
Published: September 2002

ABSTRACT

We analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of
Haemophilus
isolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the
ghfA
,
ghfD
, and
ghfE
genes previously described, we characterized two genes, designated
ghfB
and
ghfC
, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes
ghfA
to
ghfE
, homologous to genes
hifA
to
hifE
of
Haemophilus influenzae
. The sequences of the coding and intergenic regions of the
ghf
clusters of the six strains were remarkably homologous. Unlike
hif
clusters, which are inserted between
purE
and
pepN
, the
ghf
cluster was inserted between
purK
and
pepN
on the chromosome. Analysis of the flanking regions of the
ghf
cluster identified a large deletion, identical in the 5′ end regions of all strains, including the whole
purE
gene and much of the
purK
gene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that
ghf
genes were transcribed in four of the six strains. Sequencing of the intergenic
ghfA
-
ghfB
regions, including the
ghf
gene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the −10 and −35 boxes of the two overlapping and divergent promoters. The other four strains, which had
ghf
transcripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative −35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of
ghfB
induces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains.
Epitope Mapping of the Immunodominant Antigen TB10.4 and the Two Homologous Proteins TB10.3 and TB12.9, Which Constitute a Subfamily of the
esat-6
Gene Family
Authors: Skjøt Rikke Louise Vinther , Brock Inger , Arend Sandra M. , Munk Martin E. , Theisen Michael , Ottenhoff Tom H. M. , Andersen Peter 
Published: September 2002

ABSTRACT

The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the
esat
-
6
family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the
Mycobacterium tuberculosis
complex, including BCG, and
M. kansasii
, whereas other atypical mycobacteria had either one (
M. avium
,
M. intracellulare
, and
M. marinum
) or none (
M. scrofulaceum
,
M. fortuitum
, and
M. szulgai
) of the genes. The fine specificity of the T-cell response to the three closely related
esat-6
family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.
Role of RegM, a Homologue of the Catabolite Repressor Protein CcpA, in the Virulence of
Streptococcus pneumoniae
Authors: Giammarinaro Philippe , Paton James C. 
Published: September 2002

ABSTRACT

As part of a study of virulence gene regulation in
Streptococcus pneumoniae
, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins.
S. pneumoniae
D39 derivatives with insertion-duplication or deletion mutations in
regM
were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the
regM
mutants were essentially the same. However, in the presence of galactose the
regM
mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of
regM
in the catabolism of carbohydrates in
S. pneumoniae
. RegM was a repressor of both α-glucosidase and β-galactosidase activities in
S. pneumoniae
, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the
regM
mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The
regM
mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a
lacZ
transcriptional fusion construct indicated that mutation of
regM
resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (
cps
). Thus,
regM
is the first gene outside of the
cps
locus to be implicated in regulation of capsular gene expression.
Interplay between Protective and Inhibitory Antibodies Dictates the Outcome of Experimentally Disseminated Candidiasis in Recipients of aCandida albicansVaccine
Authors: Bromuro Carla , Torosantucci Antonella , Chiani Paola , Conti Stefania , Polonelli Luciano , Cassone Antonio 
Published: September 2002

ABSTRACTMice immunized with heat-inactivated, whole yeast-form cells (Y cells) ofCandida albicansdeveloped intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on wholeC. albicanscells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with β-1,3 and β-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized withC. albicanscells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candidaantibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candidaantibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.
Mycobacterium bovisBCG Vaccination Augments Interleukin-8 mRNA Expression and Protein Production in Guinea Pig Alveolar Macrophages Infected withMycobacterium tuberculosis
Authors: Lyons Mark J. , Yoshimura Teizo , McMurray David N. 
Published: September 2002

ABSTRACTAlveolar macrophages are likely the first cell type to encounterMycobacterium tuberculosisin a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated andMycobacterium bovisBCG-vaccinated guinea pigs were infected in vitro with liveM. tuberculosisH37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 μg/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuatedM. tuberculosis(H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in theM. tuberculosis-infected lung.
Influence of Intravenous Anesthesia on Mucosal and Systemic Antibody Responses to Nasal Vaccines
Authors: Janakova Libuse , Bakke Hilde , Haugen Inger Lise , Berstad Aud K. H. , Høiby E. Arne , Aaberge Ingeborg S. , Haneberg Bjørn 
Published: September 2002

ABSTRACT
Inhalation of antigens may stimulate the immune system by way of the upper as well as the lower airways. We have shown that at least 1,000 times more live pneumococci were recovered from pulmonary tissue after being presented as drops of a liquid suspension onto the nares of anesthetized mice compared to the number of bacteria recovered from animals that were not anesthetized in the course of the challenge. Mice that were similarly immunized intranasally by inhalation of three different nonreplicating particulate vaccine formulations, i.e., a meningococcal outer membrane vesicle (OMV) vaccine, a formalin-inactivated whole-virus influenza (INV) vaccine, and the INV vaccine with OMVs as a mucosal adjuvant, during general intravenous anesthesia developed concentrations of vaccine-specific serum immunoglobulin G (IgG) antibodies that were four to nine times higher than in mice that were fully awake during immunizations. The concentrations of IgA antibodies in serum were also higher in anesthetized than in nonanesthetized mice and correlated positively with the corresponding levels of serum IgG antibodies in the anesthetized but not in the nonanesthetized mice. In saliva and feces, however, the concentrations of IgA antibodies were equally high whether or not the animals were dormant during immunizations. The results indicate that intrapulmonary antigen presentation, as a part of an intranasal immunization strategy, is of importance for systemic but not for mucosal antibody responses. A major portion of IgA antibodies in serum may thus be derived from nonmucosal sites.
Purification and Characterization of a Second Immunoreactive Mannoprotein fromCryptococcus neoformansThat Stimulates T-Cell Responses
Authors: Huang Chao , Nong Shu-hua , Mansour Michael K. , Specht Charles A. , Levitz Stuart M. 
Published: September 2002

ABSTRACTAlthough T-cell responses are known to be critical for effective host defenses against the fungal pathogenCryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4+-T-cell hybridomas that react withC. neoformansantigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disruptedC. neoformansby anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search ofC. neoformansgenomic databases revealed thatMP88shares this feature with at least 11 other genes, includingMP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction ofC. neoformanssupernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.