bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

The 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

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

To 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

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

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

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

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

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.
Naturally Acquired Antibody Responses toPlasmodium falciparumMerozoite Surface Protein 4 in a Population Living in an Area of Endemicity in Vietnam
Authors: Wang Lina , Richie Thomas L. , Stowers Anthony , Nhan Doan Hanh , Coppel Ross L.
Published: July 2002

Merozoite surface protein 4 (MSP4) ofPlasmodium falciparumis a glycosylphosphatidylinositol-anchored integral membrane protein that is being developed as a component of a subunit vaccine against malaria. We report here the measurement of naturally acquired antibodies to MSP4 in a population of individuals living in the Khanh-Hoa region of Vietnam, an area where malaria is highly endemic. Antibodies to MSP4 were detected in 94% of the study population at titers of 1:5,000 or greater. Two forms of recombinant MSP4 produced in eitherEscherichia coliorSaccharomyces cerevisiaewere compared as substrates in the enzyme-linked immunosorbent assay. There was an excellent correlation between reactivity measured to either, although the yeast substrate was recognized by a higher percentage of sera. Four different regions of MSP4 were recognized by human antibodies, demonstrating that there are at least four distinct epitopes in this protein. In the carboxyl terminus, where the single epidermal growth factor-like domain is located, the reactive epitope(s) was shown to be conformation dependent, as disruption of the disulfide bonds almost completely abolished reactivity with human antibodies. The anti-MSP4 antibodies were mainly of the immunoglobulin G1 (IgG1) and IgG3 subclasses, suggesting that such antibodies may play a role in opsonization and complement-mediated lysis of free merozoites. Individuals in the study population were drug-cured and followed up for 6 months; no significant correlation was observed between the anti-MSP4 antibodies and the absence of parasitemia during the surveillance period. As a comparison, antibodies to MSP119, a leading vaccine candidate, were measured, and no correlation with protection was observed in these individuals. The anti-MSP119antibodies were predominantly of the IgG1 isotype, in contrast to the IgG3 predominance noted for MSP4.
Decreased Amounts of Cell Wall-Associated Protein A and Fibronectin-Binding Proteins in
Staphylococcus aureus sarA
Mutants due to Up-Regulation of Extracellular Proteases
Authors: Karlsson Anna , Saravia-Otten Patricia , Tegmark Karin , Morfeldt Eva , Arvidson Staffan
Published: July 2002

Data have been presented indicating that
Staphylococcus aureus
cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that in
sarA
mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation of
sarA
mutant cells in the presence of the global protease inhibitor α
2
-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in
sarA
mutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either
ssp
or
aur
in the prototype
S. aureus
strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a
sarA
mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.
Diversity in Antistaphylococcal Mechanisms among Membrane-Targeting Antimicrobial Peptides
Authors: Koo Su-Pin , Bayer Arnold S. , Yeaman Michael R.
Published: July 2002

Many antimicrobial peptides permeabilize the bacterial cytoplasmic membrane. However, it is unclear how membrane permeabilization and antimicrobial activity are related for distinct peptides. This study investigated the relationship between
Staphylococcus aureus
membrane permeabilization and cell death due to the following antistaphylococcal peptides: thrombin-induced platelet microbicidal protein 1 (tPMP-1), gramicidin D, and protamine. Isogenic
S. aureus
strains ISP479C and ISP479R (tPMP-1 susceptible and resistant, respectively), were loaded with the fluorochrome calcein and exposed to a range of concentrations of each peptide. Flow cytometry was then used to monitor membrane permeabilization by quantifying the release of preloaded calcein. Killing was determined by quantitative culture at time points simultaneous to measurement of membrane permeabilization. Membrane permeabilization and killing caused by tPMP-1 occurred in a time- and concentration-dependent manner, reflecting the intrinsic tPMP-1 susceptibilities of ISP479C and ISP479R. In comparison, gramicidin D killed both
S. aureus
strains to equivalent extents in a concentration-dependent manner between 0.5 to 50 μg/ml, but cell permeabilization only occurred at the higher peptide concentrations (25 and 50 μg/ml). Protamine permeabilized, but did not kill, either strain at concentrations up to 10 mg/ml. Regression analyses revealed different relationships between membrane permeabilization and staphylocidal activity for the distinct antimicrobial peptides. Taken together, these findings demonstrate that permeabilization, per se, does not invariably result in staphylococcal death due to distinct antimicrobial peptides. Thus, although each of these peptides interacts with the
S. aureus
cytoplasmic membrane, diversity exists in their mechanisms of action with respect to the relationship between membrane permeabilization and staphylocidal activity.
Bartonella henselaeInduces NF-κB-Dependent Upregulation of Adhesion Molecules in Cultured Human Endothelial Cells: Possible Role of Outer Membrane Proteins as Pathogenic Factors
Authors: Fuhrmann Oliver , Arvand Mardjan , Göhler Alexander , Schmid Michael , Krüll Matthias , Hippenstiel Stefan , Seybold Joachim , Dehio Christoph , Suttorp Norbert
Published: July 2002

The endothelium is a specific target forBartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms ofBartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed.B. henselaestrain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-κB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands,B. henselaeBerlin-1 was substantially more active than the typing strainB. henselaeATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-κB activation and could be blocked by a specific NF-κB inhibitor. Additional studies indicated thatB. henselae-induced effects did not require living bacteria orBartonellalipopolysaccharides. Exposure of HUVEC to purifiedB. henselaeouter membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion,B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells.B. henselaeOMPs are sufficient to induce NF-κB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties ofB. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.
Localization of Surface Immunogenic Protein on Group B Streptococcus
Authors: Rioux Stéphane , Martin Denis , Ackermann Hans-Wolfgang , Dumont Julie , Hamel Josée , Brodeur Bernard R.
Published: July 2002

The localization and accessibility of the group B streptococcus (GBS) surface immunogenic protein (Sip) at the surface of intact GBS cells were studied by flow cytometric assay and immunogold electron microscopy. Antibodies present in pooled sera collected from mice after immunization with purified recombinant Sip efficiently recognized native Sip at the surfaces of the different GBS strains tested, which included representatives of all nine serotypes. Examination of GBS cells by immunogold electron microscopy revealed that the Sip-specific antibodies attached preferentially to polar sites and the septal region. This result confirmed that Sip is exposed at the intact-cell surface, but it also suggests that its distribution is restricted to certain regions of the cell.
Novel Extracellular x-Prolyl Dipeptidyl-Peptidase (DPP) from
Streptococcus gordonii
FSS2: an Emerging Subfamily of Viridans Streptococcal x-Prolyl DPPs
Authors: Goldstein J. M. , Banbula A. , Kordula T. , Mayo J. A. , Travis J.
Published: July 2002

Streptococcus gordonii
is generally considered a benign inhabitant of the oral microflora, and yet it is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-degrading activities during growth in culture. In this report we describe the purification, characterization, and cloning of a serine class dipeptidyl-aminopeptidase, an x-prolyl dipeptidyl-peptidase (Sg-xPDPP, for
S. gordonii
x-prolyl dipeptidyl-peptidase), produced in a pH-controlled batch culture. Purification of this enzyme by anion exchange, gel filtration, and hydrophobic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. However, under native conditions, the protein appeared to be a homodimer on the basis of gel filtration and PAGE. Kinetic studies indicated that purified enzyme had a unique and stringent x-prolyl specificity that is comparable to both the dipeptidyl-peptidase IV/CD26 and lactococcal x-prolyl dipeptidyl-peptidase families. Nested PCR cloning from an
S. gordonii
library enabled the isolation and sequence analysis of the full-length gene. A 759-amino-acid polypeptide with a theoretical molecular mass of 87,115 Da and a calculated pI of 5.6 was encoded by this open reading frame. Significant homology was found with the PepX gene family from
Lactobacillus
and
Lactococcus
spp. and putative x-prolyl dipeptidyl-peptidases from other streptococcal species. Sg-xPDPP may serve as a critical factor for the sustained bacterial growth in vivo and furthermore may aid in the proteolysis of host tissue that is commonly observed during SBE pathology.
Functional Specific Binding of Testosterone toSchistosoma haematobium28-Kilodalton GlutathioneS-Transferase
Authors: Remoué Franck , Mani Jean-Claude , Pugnière Martine , Schacht Anne-Marie , Capron André , Riveau Gilles
Published: July 2002

During parasitic disease such as schistosomiasis, sex hormones have an important influence on the age- and gender-dependent level of infection. Since mammal glutathioneS-transferase (GST) has the ability to bind hormones and particularly sexual steroids to influence their transport, metabolism, and physiological action, we have evaluated the capacity of testosterone to bind the 28-kDa GST of theSchistosoma haematobiumparasite (Sh28GST). For the first time, we have demonstrated a specific binding of testosterone to parasite GST protein with high affinity (Kd= 2.57 × 10−7M). In addition, we have assessed the effect of this binding on Sh28GST enzymatic activity, a mechanism closely associated with the reduction ofSchistosomafecundity. We showed that testosterone has the functional ability to inhibit the Sh28GST enzymatic activity in a dose-dependent manner, suggesting that this hormone could be directly involved in an antifecundity mechanism. This effect seemed to be related to the binding of testosterone to one peptide involved in the enzymatic site (i.e., amino acids 24 to 43). During human infection, binding of sexual hormones toSchistosomaSh28GST could play a key role in parasite metabolism, especially the decrease of fecundity, and could be involved in the sex-dependent immune response to Sh28GST that we have previously observed in infected adults.
Control of ExperimentalTrypanosoma bruceiInfections Occurs Independently of Lymphotoxin-α Induction
Authors: Magez S. , Stijlemans B. , Caljon G. , Eugster H.-P. , De Baetselier P.
Published: July 2002

Trypanosome infections are marked by severe pathological features, including anemia, splenomegaly, and suppression of T-cell proliferation. We have used lymphotoxin-α-deficient (LT-α−/−) mice, as well as LT-α-tumor necrosis factor-double-deficient (LT-α−/−TNF−/−) mice, to analyze the contributions of these related cytokines in both induction of trypanosomosis-associated immunopathology and infection control. Moreover, as the cytokine-deficient mice used have no detectable lymph nodes and lack germinal-center formation upon immune stimulation, we have analyzed the functional importance of both the lymph nodes and spleen during experimentalTrypanosoma bruceiinfections. First, we show that the absence of LT-α does not significantly alter early trypanosomosis development or pathology but does result in better control of late-stage parasitemia levels and slightly prolonged survival. This increased survival of infected LT-α−/−mice coincides with the appearance of increased chronic-stage anti-trypanosome immunoglobulin M (IgM)-IgG2a serum titers that are generated in the absence of functional peripheral lymphoid tissue and do not require germinal-center formation. Second, we show that splenectomized mice control their parasitemia to the same extent as fully immune-competent littermates. Finally, using LT-α−/−TNF−/−double-deficient mice, we show that in these miceT. bruceiinfections are very well controlled during the chronic infection stage and that infection-induced pathology is minimized. Together, these findings indicate that while increased IgM-IgG2a anti-trypanosome antibody titers (generated in the absence of LT-α, peripheral lymph nodes, and germinal-center formation) coincide with improved parasitemia control, it is TNF that has a major impact on trypanosomosis-associated immunopathology.
Survival ofTropheryma whipplei, the Agent of Whipple's Disease, Requires Phagosome Acidification
Authors: Ghigo Eric , Capo Christian , Aurouze Marianne , Tung Ching-Hsuan , Gorvel Jean-Pierre , Raoult Didier , Mege Jean-Louis
Published: July 2002

Tropheryma whippleiwas established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here thatT. whippleisurvives within HeLa cells by controlling the biogenesis of its phagosome. Indeed,T. whippleicolocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition,T. whippleisurvived within HeLa cells by adapting to acidic pH. The vacuoles containingT. whippleiwere acidic (pH 4.7 ± 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride,N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing ofT. whipplei. The ability ofT. whippleito survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.
Deletion of T Cells Bearing the Vβ8.1 T-Cell Receptor following Mouse Mammary Tumor Virus 7 Integration Confers Resistance to Murine Cerebral Malaria
Authors: Gorgette Olivier , Existe Alexandre , Boubou Mariama Idrissa , Bagot Sébastien , Guénet Jean-Louis , Mazier Dominique , Cazenave Pierre-André , Pied Sylviane
Published: July 2002

Plasmodium bergheiANKA induces a fatal neurological syndrome known as cerebral malaria (CM) in susceptible mice. Host genetic elements are among the key factors determining susceptibility or resistance to CM. Analysis of mice of the sameH-2haplotype revealed that mouse mammary tumor virus 7 (MTV-7) integration into chromosome 1 is one of the key factors associated with resistance to neurological disease duringP. bergheiANKA infection. We investigated this phenomenon by infecting a series of recombinant inbred mice (CXD2), derived from BALB/c (susceptible to CM) and DBA/2 (resistant to CM) mice, withP. bergheiANKA. We observed differences in susceptibility to CM induced by thisPlasmodiumstrain. Mice with the MTV-7 sequence in their genome were resistant to CM, whereas those without integration of this gene were susceptible. Thus, an integrated proviral open reading frame or similar genomic sequences may confer protection against neuropathogenesis during malaria, at least in mice.
Treponema denticolaIs Resistant to Human β-Defensins
Authors: Brissette Catherine A. , Lukehart Sheila A.
Published: July 2002

Spirochetes, includingTreponema denticola, are implicated in the pathogenesis of periodontal disease. BecauseT. denticolalacks lipopolysaccharides that serve as targets for human β-defensin (hβD) binding, we postulated thatT. denticolawould resist killing by hβD. We showed thatT. denticolais resistant to hβD-1 and -2. Protease inhibitors did not enhance killing ofT. denticolaby hβD-2, suggesting that degradation of hβD-2 by treponemal proteases is not a major factor inT. denticolaresistance.
FabG, an NADPH-Dependent 3-Ketoacyl Reductase ofPseudomonas aeruginosa, Provides Precursors for Medium-Chain-Length Poly-3-Hydroxyalkanoate Biosynthesis inEscherichia coli
Authors: Ren Qun , Sierro Nicolas , Witholt Bernard , Kessler Birgit
Published: July 2002

Escherichia colihosts expressingfabGofPseudomonas aeruginosashowed 3-ketoacyl coenzyme A (CoA) reductase activity towardR-3-hydroxyoctanoyl-CoA. Furthermore,E. colirecombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding genephaCin addition tofabGaccumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. WhenE. coli fadBorfadAmutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during β-oxidation, respectively, were transformed withfabG, higher levels of PHA were synthesized inE. coli fadA, whereas similar levels of PHA were found inE. coli fadB, compared with those of the corresponding mutants carryingphaCalone. These results strongly suggest that FabG ofP. aeruginosais able to reduce mcl-3-ketoacyl-CoAs generated by the β-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.
Proteolysis of Bacteriophage λ CII by
Escherichia coli
FtsH (HflB)
Authors: Shotland Yoram , Shifrin Amir , Ziv Tamar , Teff Dinah , Koby Simi , Kobiler Oren , Oppenheim Amos B.
Published: July 2002

FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage λ lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the λ CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage λ CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. β-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.
Hypersensitivity of
Escherichia coli Δ
(
uvrB-bio
) Mutants to 6-Hydroxylaminopurine and Other Base Analogs Is Due to a Defect in Molybdenum Cofactor Biosynthesis
Authors: Kozmin Stanislav G. , Pavlov Youri I. , Dunn Ronnie L. , Schaaper Roel M.
Published: July 2002

We have shown previously that
Escherichia coli
and
Salmonella enterica
serovar Typhimurium strains carrying a deletion of the
uvrB-bio
region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the
uvrB
excision repair defect associated with this deletion because a
uvrB
point mutation or a
uvrA
deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the
moeA
gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the
uvrB-bio
deletion. The
moeAB
operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as
moa
or
mod
, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.
Requirements for Conversion of the Na
+
-Driven Flagellar Motor of
Vibrio cholerae
to the H
+
-Driven Motor of
Escherichia coli
Authors: Gosink Khoosheh K. , Häse Claudia C.
Published: July 2002

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H
+
or Na
+
into mechanical work. In
Escherichia coli
, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of
Vibrio
species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in
pomA
,
pomB
,
motX
, or
motY
in
Vibrio cholerae
resulted in a nonmotile phenotype, whereas deletion of
fliG
gave a nonflagellate phenotype.
fliG
genes on plasmids complemented
fliG
-null strains of the parent species but not
fliG
-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A
V. cholerae
strain deleted of
pomA
,
pomB
,
motX
, and
motY
became weakly motile when the
E. coli motA
and
motB
genes were introduced on a plasmid. Like
E. coli
, but unlike wild-type
V. cholerae
, motility of some
V. cholerae
strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide
m
-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the
E. coli
proton motor components MotA and MotB can function in place of the motor proteins of
V. cholerae
and that the hybrid motors are driven by the proton motive force.
Identification and Characterization of Genes Required for Early
Myxococcus xanthus
Developmental Gene Expression
Authors: Guo Dongchuan , Wu Yun , Kaplan Heidi B.
Published: July 2002

Starvation and cell density regulate the developmental expression of
Myxococcus xanthus
gene
4521
. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn
5 lac
Ω4521 fusion are Lac
+
. One class of these mutants inactivates SasN, a negative regulator of
4521
expression; another class activates SasS, a sensor kinase-positive regulator of
4521
expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of
4521
expression, 11 Lac
−
Tn
V.AS
transposon insertion mutants were isolated from a screen of 18,000 Lac
+
LPS O-antigen mutants containing Tn
5 lac
Ω4521 (Tc
r
). Ten mutations identified genes that could encode positive regulators of
4521
developmental expression based on their ability to abolish
4521
expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the
sasB
locus, which encodes the known
4521
regulators SasS and SasN. One mapped to
sasS
, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated
sasR
, and four others mapped to a gene designated
sasP
. One mutation, designated
ssp10
, specifically suppressed the LPS O-antigen defect; the
ssp10
mutation had no effect on
4521
expression in an otherwise wild-type background but reduced
4521
developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in
sasP
conferred defects during growth and development. These data indicate that a number of elements are required for
4521
developmental expression and that most of these are necessary for normal growth and fruiting body development.
Effects of the Calvin Cycle on Nicotinamide Adenine Dinucleotide Concentrations and Redox Balances of
Xanthobacter flavus
Authors: van Keulen Geertje , Dijkhuizen Lubbert , Meijer Wim G.
Published: July 2002

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in
Xanthobacter flavus
during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.
Biochemical and Molecular Characterization of Phenylacetate-Coenzyme A Ligase, an Enzyme Catalyzing the First Step in Aerobic Metabolism of Phenylacetic Acid in
Azoarcus evansii
Authors: El-Said Mohamed Magdy
Published: July 2002

Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC
6.2.1.30
), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in
Azoarcus evansii
, has been purified and characterized. The gene (
paaK
) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min
−1
mg of cell protein
−1
was measured. After 117-fold purification to homogeneity, a specific activity of 48 μmol min
−1
mg of protein
−1
was achieved with a turnover number (catalytic constant) of 40 s
−1
. The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent

K
m

values for PA, ATP, and CoA were 14, 60, and 45 μM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1,320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium.