bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the
Helicobacter pylori ureI
Gene Is Required for Acidic pH Activation of Cytoplasmic Urease
Authors: Scott David R. , Marcus Elizabeth A. , Weeks David L. , Lee Adrian , Melchers Klaus , Sachs George 
Published: July 2002

ABSTRACT

ureI
encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of
Helicobacter pylori
and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced
H. pylori ureI
deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined
ureI
expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric
Helicobacter
species.
ureI
was found to be present in the genome of the gastric
Helicobacter
species and absent in the nongastric
Helicobacter
species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric
Helicobacter
species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric
Helicobacter
species. The
ureI
gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.
A Model of Infected Burn Wounds Using
Escherichia coli
O18:K1:H7 for the Study of Gram-Negative Bacteremia and Sepsis
Authors: Busch Nathan A. , Zanzot Emily M. , Loiselle Paul M. , Carter Edward A. , Allaire Jennifer E. , Yarmush Martin L. , Warren H. Shaw 
Published: July 2002

ABSTRACT

A difficulty that has emerged in the development and preclinical evaluation of adjuvant therapies for gram-negative sepsis is the lack of easily studied animal models that closely mimic human infection. An objective of this study was to adapt a previously described model of infection in burned mice to rats with a defined bacterial strain of
Escherichia coli
. Challenge with two colonies of live
E. coli
O18:K1:H7 bacteria into an 8% full-thickness burn of the dorsal skin surface of rats produced predictable bacteremia at 24 to 48 h and 80 to 100% mortality at 3 to 4 days.
E. coli
O18:K1:H7 was approximately 10-million-fold more virulent than several other gram-negative bacterial strains. The model should be a useful tool in studying the pathogenicity of burn wound infections and in evaluating the efficacy of novel adjuvant therapies for gram-negative sepsis.
Coxiella burnetii
Exhibits Morphological Change and Delays Phagolysosomal Fusion after Internalization by J774A.1 Cells
Authors: Howe Dale , Mallavia Louis P. 
Published: July 2002

ABSTRACT

Coxiella burnetii
, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in
C. burnetii
survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization of
C. burnetii
by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [
35
S]methionine-[
35
S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake
C. burnetii
was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable
C. burnetii
was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.
Characterization of a Shiga Toxin 2e-Converting Bacteriophage from an
Escherichia coli
Strain of Human Origin
Authors: Muniesa Maite , Recktenwald Jürgen , Bielaszewska Martina , Karch Helge , Schmidt Herbert 
Published: July 2002

ABSTRACT

An infectious Shiga toxin (Stx) 2e-converting bacteriophage (φP27) was isolated from Stx2e-producing
Escherichia coli
ONT:H
−
isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to
E. coli
laboratory strain DH5α, and we could show that lysogens were able to produce biologically active toxin in a
recA
-dependent manner. By DNA sequence analysis of a 6,388-bp
Hin
dIII restriction fragment of φP27, we demonstrated that the
stx
2e
gene was located directly downstream of
ileZ
and
argO
tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of
Pseudomonas aeruginosa
phage φCTX and a gene homologous to the endolysin gene
gp19
of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.
Diversity of
ace
, a Gene Encoding a Microbial Surface Component Recognizing Adhesive Matrix Molecules, from Different Strains of
Enterococcus faecalis
and Evidence for Production of Ace during Human Infections
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Duh Ruay-Wang , Weinstock George M. , Murray Barbara E. 
Published: July 2002

ABSTRACT

Our previous work reported that most
Enterococcus faecalis
strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46°C, but not 37°C, and we subsequently identified an
E. faecalis
sequence,
ace
, that encodes a bacterial adhesin similar to the collagen binding protein Cna of
Staphylococcus aureus
. In this study, we examined the diversity of
E. faecalis
-specific
ace
gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine
E. faecalis
strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5′ region of
ace
that encodes the A domain was sequenced, and these sequences showed ≥97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of
S. aureus
, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from
E. faecalis
strain OG1RF detected Ace in mutanolysin extracts of seven of nine
E. faecalis
strains after growth at 46°C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with
E. faecalis
endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other
E. faecalis
antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an
E. faecalis
endocarditis patient serum inhibited adherence of 46°C-grown
E. faecalis
OG1RF to collagen types I and IV and laminin. In conclusion, these results show that
ace
is highly conserved among isolates of
E. faecalis
, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.
Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion
Authors: Spyres Lea M. , Qa'Dan Maen , Meader Amy , Tomasek James J. , Howard Eric W. , Ballard Jimmy D. 
Published: July 2002

ABSTRACT

TcdB from
Clostridium difficile
glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB
1-556
coding region were constructed, expressed, and purified from
Escherichia coli
. LFnTcdB
1-556
was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB
1-556
plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.
Time between Inoculations and Karyotype Forms ofPneumocystis cariniif. sp.cariniiInfluence Outcome of Experimental Coinfections in Rats
Authors: Cushion Melanie T. , Orr Sally , Keely Scott P. , Stringer James R. 
Published: July 2002

ABSTRACTThe prevalence ofPneumocystis cariniipneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP.P. cariniif. sp.cariniiinfecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans.P. cariniif. sp.cariniikaryotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections andP. cariniif. sp.cariniikaryotype profiles were associated with differences in biological responses. This model provides a useful method for the study ofP. cariniicoinfections and their transmission in humans.
Silencing of Oxidative Stress Response in
Mycobacterium tuberculosis
: Expression Patterns of
ahpC
in Virulent and Avirulent Strains and Effect of
ahpC
Inactivation
Authors: Springer B. , Master S. , Sander P. , Zahrt T. , McFalone M. , Song J. , Papavinasasundaram K. G. , Colston M. J. , Boettger E. , Deretic V. 
Published: July 2002

ABSTRACT

Intracellular pathogens such as
Mycobacterium tuberculosis
are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (
ahpC
) of
M. tuberculosis
is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations,
ahpC
expression in virulent strains of
M. tuberculosis
and
Mycobacterium bovis
grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain
M. bovis
BCG is constitutively high. The repression of the
ahpC
gene of the virulent strains is independent of the naturally occurring lesions of central regulator
oxyR.
Using a green fluorescence protein vector (
gfp
)-
ahpC
reporter construct we present data showing that repression of
ahpC
of virulent
M. tuberculosis
also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of
ahpC
on the chromosome of
M. tuberculosis
by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H
2
O
2
. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the
ahpC
::Km
r
mutant strain. The preservation of a functional
ahpC
gene in
M. tuberculosis
in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of
ahpC
expression in vitro was lifted under conditions of static growth.
Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) ofPorphyromonasgingivalisin a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant
Authors: Yagishita Hisao , Kumagai Yumi , Konishi Kiyoshi , Takahashi Yukihiro , Aoba Takaaki , Yoshikawa Masanosuke 
Published: July 2002

ABSTRACTTo elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence ofPorphyromonasgingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells.
Phase Variation in the
Helicobacter pylori
Phospholipase A Gene and Its Role in Acid Adaptation
Authors: Tannæs Tone , Dekker Niek , Bukholm Geir , Bijlsma Jetta J. E. , Appelmelk Ben J. 
Published: July 2002

ABSTRACT

Previously, we have shown that
Helicobacter pylori
can spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The “lyso” variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the “normal” variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the
pldA
gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncated
pldA
, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in the
pldA
gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of
H. pylori
from patients referred to gastroscopy for dyspepsia.
Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of
Shigella sonnei
Authors: Strauch Eckhard , Lurz Rudi , Beutin Lothar 
Published: July 2002

ABSTRACT

A Shiga toxin (Stx)-encoding temperate bacteriophage of
Shigella sonnei
strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization in
Shigella
and
Escherichia coli
strains. Phage 7888 formed plaques on a broad spectrum of
Shigella
strains belonging to different species and serotypes, including Stx-producing
Shigella dysenteriae
type 1. With
E. coli
, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic
S. sonnei
and laboratory
E. coli
K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in
E. coli
K-12. The relationships of phage 7888 with the
E. coli
Stx1-producing phage H-19B and the
E. coli
Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the
stx
genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of
stxA
and
stxB
(
stx
gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that
stx
genes are naturally transferable and are expressed in strains of
S. sonnei
, which points to the continuous evolution of human-pathogenic
Shigella
by horizontal gene transfer.
Amino-Terminal Hydrophobic Region of
Helicobacter pylori
Vacuolating Cytotoxin (VacA) Mediates Transmembrane Protein Dimerization
Authors: McClain Mark S. , Cao Ping , Cover Timothy L. 
Published: July 2002

ABSTRACT

Helicobacter pylori
VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells. A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences. To examine functional properties of this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W. P. Russ and D. M. Engelman (Proc. Natl. Acad. Sci. USA 96:863–868, 1999) to study transmembrane helix-helix associations in a natural membrane environment. A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of
Escherichia coli
and mediated protein dimerization. A fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 of VacA was replaced by alanine) also inserted into the inner membrane but dimerized significantly less efficiently than the fusion protein containing the wild-type VacA sequence. Based on these results, we speculate that the wild-type VacA amino-terminal hydrophobic region contributes to oligomerization of the toxin within membranes of eukaryotic cells.
Comparison of the Hydrophobic Properties of
Candida albicans
and
Candida dubliniensis
Authors: Hazen Kevin C. , Wu Jean G. , Masuoka James 
Published: July 2002

ABSTRACT

Although
Candida dubliniensis
is a close genetic relative of
Candida albicans
, it colonizes and infects fewer sites. Nearly all instances of candidiasis caused by
C. dubliniensis
are restricted to the oral cavity. As cell surface hydrophobicity (CSH) influences virulence of
C. albicans
, CSH properties of
C. dubliniensis
were investigated and compared to
C. albicans
. Growth temperature is one factor which affects the CSH status of stationary-phase
C. albicans
. However,
C. dubliniensis
, similar to other pathogenic non-albicans species of
Candida
, was hydrophobic regardless of growth temperature. For all
Candida
species tested in this study (
C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis
, and
C. tropicalis
), CSH status correlated with coaggregation with the anaerobic oral bacterium
Fusobacterium nucleatum
. Previous studies have shown that CSH status of
C. albicans
involves multiple surface proteins and surface protein N-glycans. The hydrophobic surface glycoprotein CAgp38 appears to be expressed by
C. albicans
constitutively regardless of growth temperature and medium.
C. dubliniensis
expresses a 38-kDa protein that cross-reacts with the anti-CAgp38 monoclonal antibody; however, expression of the protein was growth medium and growth temperature dependent. The anti-CAgp38 monoclonal antibody has been shown to inhibit adhesion of
C. albicans
to extracellular matrix proteins and to vascular endothelial cells. Since protein glycosylation influences the CSH status of
C. albicans
, we compared the cell wall mannoprotein content and composition between
C. albicans
and
C. dubliniensis
. Similar bulk compositional levels of hexose, phosphate, and protein in their N-glycans were determined. However, a component of the
C. albicans
N-glycan, acid-labile phosphooligomannoside, is expressed much less or negligibly by
C. dubliniensis
, and when present, the oligomannosides are predominantly less than five mannose residues in length. In addition, the acid-labile phosphooligomannoside profiles varied among the three strains of
C. dubliniensis
we tested, indicating the N-glycan of
C. dubliniensis
differs from
C. albicans
. For
C. albicans
, the acid-labile phosphooligomannoside influences virulence and surface fibrillar conformation, which affects exposure of hydrophobic surface proteins. Given the combined role in
C. albicans
of expression of specific surface hydrophobic proteins in pathogenesis and of surface protein glycosylation on exposure of the proteins, the lack of these virulence-associated CSH entities in
C. dubliniensis
could contribute to its limited ability to cause disseminated infections.
Genetic Loci of
Streptococcus mitis
That Mediate Binding to Human Platelets
Authors: Bensing Barbara A. , Rubens Craig E. , Sullam Paul M. 
Published: July 2002

ABSTRACT

The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis. To identify bacterial components that mediate platelet binding by
Streptococcus mitis
, we screened a Tn
916
ΔE-derived mutant library of
S. mitis
strain SF100 for reduced binding to human platelets in vitro. Two distinct loci were found to affect platelet binding. The first contains a gene (
pblT
) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments. This protein resembles members of the major facilitator superfamily of small-molecule transporters. The second platelet binding locus consists of an apparent polycistronic operon. This region includes genes that are highly similar to those of
Lactococcus lactis
phage r1t and
Streptococcus thermophilus
phage 01205. Two genes (
pblA
and
pblB
) encoding large surface proteins are also present. The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall. The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205. Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA. The combined data indicate that at least two genomic regions contribute to platelet binding by
S. mitis.
One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.