bluuebunny/crossref_metadata_2025_split huggingface dataset:
Superoxide Dismutase Influences the Virulence ofCryptococcus neoformansby Affecting Growth within Macrophages
Authors: Cox Gary M. , Harrison Thomas S. , McDade Henry C. , Taborda Carlos P. , Heinrich Garrett , Casadevall Arturo , Perfect John R. 
Published: December 2002

ABSTRACTSuperoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeastCryptococcus neoformansand made mutants via targeted disruption. Thesod1mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. Asod1mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that thesod1strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity inC. neoformans; (ii) the decreased virulence of thesod1strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems inC. neoformanscan compensate for the loss of the Cu,Zn SOD in vivo.
Purification and Characterization of a UDP-Glucosyltransferase Produced by
Legionella pneumophila
Authors: Belyi Iouri , Popoff Michel R. , Cianciotto Nicholas P. 
Published: December 2002

ABSTRACT

Legionella pneumophila
is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the
Legionella
interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of
L. pneumophila
, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from
L. pneumophila
and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished
L. pneumophila
genome database. The intact gene was cloned and expressed in
Escherichia coli
, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene
ugt
(
U
DP-glucosyl
t
ransferase). The
Legionella
enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in
L. pneumophila
strains but was absent from the other
Legionella
species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote
L. pneumophila
proliferation.
Molecular Heterogeneity of
Ehrlichia chaffeensis
Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome
Authors: Cheng Chuanmin , Paddock Christopher D. , Reddy Ganta Roman 
Published: December 2002

ABSTRACT

Ehrlichia chaffeensis
, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene deletion. There were two regions on the locus where novel gene deletion or insertion mutations occurred, resulting in the net loss of one gene in Group II isolates. Numerous nucleotide differences among genes in isolates of each group also were detected. The shared homology among isolates in each group for the 28-kDa OMP locus suggests the derivation of clonal lineages. Transcription and translation analysis of the locus revealed differences in the expressed genes of different group isolates. Analysis of the 120-kDa OMP gene and variable-length PCR target gene showed size variations resulting from loss or gain of long, direct repeats within the protein coding sequences. To our knowledge this is the first study that looked at several regions of the genome simultaneously, and we provide the first evidence of heterogeneity resulting from gene deletion and insertion mutations in the
E. chaffeensis
genome. Diversity in different genomic regions could be the result of a selection process or of independently evolved genes.
Phagocyte NADPH Oxidase, but Not Inducible Nitric Oxide Synthase, Is Essential for Early Control of
Burkholderia cepacia
and
Chromobacterium violaceum
Infection in Mice
Authors: Segal Brahm H. , Ding Li , Holland Steven M. 
Published: December 2002

ABSTRACT

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47

phox

−/−
) and mice deficient in generating inducible nitric oxide synthase (iNOS
−/−
), we examined the roles of these reactive species in host defense against
Burkholderia cepacia
and
Chromobacterium violaceum
, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal
B. cepacia
challenge (4.0 × 10
3
to 4.0 × 10
5
organisms/mouse) resulted in mortality in all p47

phox

−/−
mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival.
C. violaceum
was strikingly virulent in p47

phox

−/−
mice (the 50% lethal dose [LD
50
] was <13 organisms). iNOS
−/−
and wild-type mice were resistant to
B. cepacia
challenges of at least 10
6
organisms per mouse, and the LD
50
of
C. violaceum
was between 10
6
and 10
7
organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of
B. cepacia
from multiple sites were higher for p47

phox

−/−
mice than for iNOS
−/−
and wild-type mice at day 4 after challenge, but numbers of organisms for different
B. cepacia
strains varied. The recovery of
C. violaceum
was strikingly greater at 18 h after challenge for p47

phox

−/−
mice than for iNOS
−/−
and wild-type mice, in which the organism burdens were virtually nil. In vitro, both
B. cepacia
and
C. violaceum
were sensitive to H
2
O
2
and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against
B. cepacia
and
C. violaceum
is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.
Identification of Substrates and Chaperone from the
Yersinia enterocolitica
1B Ysa Type III Secretion System
Authors: Foultier Boris , Troisfontaines Paul , Vertommen Didier , Marenne Marie-Noëlle , Rider Mark , Parsot Claude , Cornelis Guy R. 
Published: December 2002

ABSTRACT

All pathogenic
Yersinia enterocolitica
strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37°C. In addition, biovar 1B
Y. enterocolitica
strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames,
sycB
,
yspB
,
yspC
,
yspD
,
yspA
, and
acpY
, neighbor the
ysa
genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of
Shigella flexneri
and SPI-1 of
Salmonella enterica
. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both
Y. enterocolitica
TTS systems. YspC could also be secreted by
S. flexneri
, but no functional complementation of
ipaC
was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the
yopE
promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.
THP-1 Cell Apoptosis in Response to Mycobacterial Infection
Authors: Riendeau Carrie J. , Kornfeld Hardy 
Published: December 2002

ABSTRACTWe previously reported thatMycobacterium tuberculosisinfection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-α)-mediated apoptosis and that macrophage apoptosis is associated with killing internalized bacilli. Virulent mycobacterial strains elicit much less apoptosis than attenuated strains, implying that apoptosis is a defense against intracellular infection. The present study evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells to mimic this response of primary macrophages. Consistent with the behavior of alveolar macrophages, attenuatedM. tuberculosisH37Ra andMycobacterium bovisBCG strongly induce THP-1 apoptosis, which requires endogenous TNF. THP-1 apoptosis is associated with reduced viability of infecting BCG. In contrast, virulent wild-typeM. tuberculosisH37Rv andM. bovisdo not increase THP-1 apoptosis over baseline. BCG induced early activation of caspase 10 and 9, followed by caspase 3. In contrast, wild-typeM. bovisinfection failed to activate any caspases in THP-1 cells. BCG-induced THP-1 apoptosis is blocked by retroviral transduction with vectors expressingcrmAbut notbcl-2. We conclude that differentiated THP-1 cells faithfully model the apoptosis response of HAM. Analysis of the THP-1 cell response to infection with virulent mycobacteria suggests that TNF death signals are blocked proximal to initiator caspase activation, at the level of TNF receptor 1 or its associated intracytoplasmic adaptor complex. Interference with TNF death signaling may be a virulence mechanism that allowsM. tuberculosisto circumvent innate defenses leading to apoptosis of infected host cells.
Entry of the Lymphogranuloma Venereum Strain ofChlamydia trachomatisinto Host Cells Involves Cholesterol-Rich Membrane Domains
Authors: Jutras Isabelle , Abrami Laurence , Dautry-Varsat Alice 
Published: December 2002

ABSTRACTChlamydiae are bacterial pathogens which develop strictly inside the epithelial cells of their hosts. The mechanism used by chlamydiae to enter cells is not well characterized; however, it is thought to consist of a receptor-mediated process. In addition, the formation of clathrin-coated pits appears to be dispensable for chlamydiae to be internalized by host cells. Clathrin-independent endocytosis has recently been shown to occur through cholesterol-rich lipid microdomains, which are characterized by detergent insolubility. In the present study, we investigated whether these lipid domains play a role inChlamydia trachomatisserovar L2 internalization by host cells. Our results show that after binding to HeLa cells, chlamydiae are associated with detergent-resistant lipid microdomains (DRMs), which can be isolated by fractionation of infected HeLa cells and flotation on a sucrose gradient. After internalization by HeLa cells, chlamydiae were still found in DRMs. In addition, extraction of plasma membrane cholesterol inhibited infection of HeLa cells byC. trachomatis. Many of the proteins associated with DRMs are glycosylphosphatidylinositol (GPI)-anchored proteins; however, our results could not identify a role for GPI-anchored proteins in the entry process. The same results were obtained forChlamydia psittacistrain GPIC. We propose that cholesterol-rich domains participate in the entry of chlamydiae into host cells.Chlamydiabinding to cholesterol-rich domains may lead to coalescence of the bacterial cells, which could trigger internalization by host cells.
Interleukin-1 Receptor Antagonist Gene Polymorphism, Vaginal Interleukin-1 Receptor Antagonist Concentrations, and VaginalUreaplasma urealyticumColonization in Pregnant Women
Authors: Barton Parrin T. , Gerber Stefan , Skupski Daniel W. , Witkin Steven S. 
Published: December 2002

ABSTRACTUreaplasma urealyticumis the microorganism most frequently isolated from amniotic fluids of women in preterm labor. The relationship between vaginal colonization withU. urealyticum,vaginal interleukin-1 receptor antagonist (IL-1ra) levels, and the IL-1ra genotype in pregnant women was examined. Vaginal specimens, obtained with a cotton swab from 207 women in their first trimester of pregnancy, were tested for IL-1ra concentrations by enzyme-linked immunosorbent assay and forU. urealyticumand IL-1ra genotypes by PCR.U. urealyticumwas detected in 85 (41.1%) women. The median IL-1ra level was 450 ng/ml in women positive forU. urealyticum, as opposed to 225 ng/ml in women negative for this microorganism (P< 0.0001). Sixty-two percent of the 16 women who were homozygous for allele 2 of the IL-1ra gene (IL-1RN*2) were colonized withU. urealyticum, as opposed to 47% of the 49 women who wereIL-1RN*1/IL-1RN*2heterozygotes and 34% of the 133 women who wereIL-1RN*1homozygotes (P< 0.05). Median IL-1ra levels were 750 ng/ml inIL-1RN*2homozygotes, 300 ng/ml inIL-1RN*1/IL-1RN*2heterozygotes, and 250 ng/ml inIL-1RN*1homozygotes (P= 0.02). The vast majority of subjects had an uneventful pregnancy and delivered a healthy infant at term. The IL-1ra genotype orU. urealyticumcolonization was unrelated to birth weight. Pregnant women who are colonized withU. urealyticumduring the first trimester have elevated vaginal IL-1ra concentrations and a higher prevalence of theIL-1RN*2homozygote genotype than do noncolonized women.
Oral Immunization with ATP-Dependent Protease-Deficient Mutants Protects Mice against Subsequent Oral Challenge with VirulentSalmonella entericaSerovar Typhimurium
Authors: Matsui Hidenori , Suzuki Masato , Isshiki Yasunori , Kodama Chie , Eguchi Masahiro , Kikuchi Yuji , Motokawa Kenji , Takaya Akiko , Tomoyasu Toshifumi , Yamamoto Tomoko 
Published: December 2002

ABSTRACTWe evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains againstSalmonellainfection through infection studies with mice by using aSalmonella entericaserovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol.184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol.184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 × 108CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 103to 104CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate thatSalmonellawith a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.
A GRA1 DNA Vaccine Primes Cytolytic CD8+T Cells To Control AcuteToxoplasma gondiiInfection
Authors: Scorza T. , D'Souza S. , Laloup M. , Dewit J. , De Braekeleer J. , Verschueren H. , Vercammen M. , Huygen K. , Jongert E. 
Published: December 2002

ABSTRACTProtective immunity againstToxoplasma gondiiis known to be mediated mainly by T lymphocytes and gamma interferon (IFN-γ). The contribution of CD4+and CD8+T-lymphocyte subsets to protective immune responses againstT. gondiiinfection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4+and CD8+T-cell subsets produced IFN-γ upon restimulation with aT. gondiilysate. In addition, the GRA1 DNA vaccine elicited CD8+T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge withT. gondiicysts. In vivo T-cell depletion experiments indicated that CD8+T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase ofT. gondiiinfection, while depletion of CD4+T cells led to an increase in brain cyst burden during the chronic phase of infection.
Role for Recombinant γ-Glutamyltransferase from
Treponema denticola
in Glutathione Metabolism
Authors: Chu Lianrui , Xu Xiaoping , Dong Zheng , Cappelli David , Ebersole Jefferey L. 
Published: December 2002

ABSTRACT

Volatile sulfur compounds, including hydrogen sulfide (H
2
S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H
2
S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in
Treponema denticola
that releases H
2
S. In this pathway, γ-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from
T. denticola
, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into
Escherichia coli
led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-γ-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine.
l
-Cysteine is not a substrate of GGT. Importantly, GNA, when added to
T. denticola
, was able to compete with glutathione and inhibit the production of H
2
S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (
N
α-
p
-tosyl-
l
-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to
Porphyromonas gingivalis
, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H
2
S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.
sarU
, a
sarA
Homolog, Is Repressed by SarT and Regulates Virulence Genes in
Staphylococcus aureus
Authors: Manna Adhar C. , Cheung Ambrose L. 
Published: December 2002

ABSTRACT

In searching the
Staphylococcus aureus
genome, we previously identified
sarT
, a homolog of
sarA,
which encodes a repressor for α-hemolysin synthesis. Adjacent but transcribed divergently to
sarT
is
sarU,
which encodes a 247-residue polypeptide, almost twice the length of SarA. Sequence alignment disclosed that SarU, like SarS, which is another SarA homolog, could be envisioned as a molecule with two halves, with each half being homologous to SarA. SarU, as a member of the SarA family proteins, disclosed conservation of basic residues within the helix-turn-helix motif and within the beta hairpin loop, two putative DNA binding domains within this protein family. The transcription of
sarU
is increased in a
sarT
mutant. Gel shift and transcriptional fusion studies revealed that SarT can bind to the
sarU
promoter region, probably acting as a repressor for
sarU
transcription. The expression of RNAII and RNAIII of
agr
is decreased in a
sarU
mutant. As RNAIII expression is up-regulated in a
sarT
mutant, we hypothesize that
sarT
may down regulate
agr
RNAIII expression by repressing
sarU
, a positive activator of
agr
expression. We propose that, in addition to the quorum sensing effect of the autoinducing peptide of
agr
, the
sarT
-
sarU
pathway may represent a secondary amplification loop whereby the expression of
agr
(e.g., those found in vivo) might repress
sarT
, leading to increased expression of
sarU
. Elevated
sarU
expression would result in additional amplification of the original
agr
signal.
Differential Expression of Gamma Interferon mRNA Induced by Attenuated and VirulentMycobacterium tuberculosisin Guinea Pig Cells afterMycobacterium bovisBCG Vaccination
Authors: Jeevan Amminikutty , Yoshimura Teizo , Lee Kyeong Eun , McMurray David N. 
Published: December 2002

ABSTRACTTo determine whetherMycobacterium bovisBCG vaccination would alter gamma interferon (IFN-γ) mRNA expression in guinea pig cells exposed toMycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-γ from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-γ, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) orM. tuberculosisH37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a32P-labeled probe derived from the cDNA clone. Compared to the IFN-γ mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-γ mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulentM. tuberculosisbacterium per 10 cells. The enhanced IFN-γ mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4+T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-γ mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-γ mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-γ mRNA accumulation is currently under investigation.