bluuebunny/crossref_metadata_2025_split huggingface dataset:
The DnaK/DnaJ Chaperone Machinery of
Salmonella enterica
Serovar Typhimurium Is Essential for Invasion of Epithelial Cells and Survival within Macrophages, Leading to Systemic Infection
Authors: Takaya Akiko , Tomoyasu Toshifumi , Matsui Hidenori , Yamamoto Tomoko 
Published: February 2004

ABSTRACT

Salmonella enterica
serovar Typhimurium, similar to various facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by inducing stress proteins, such as DnaK. DnaK forms a chaperone machinery with the cochaperones DnaJ and GrpE. To elucidate the role of the DnaK chaperone machinery in the pathogenesis of
S. enterica
serovar Typhimurium, we first constructed an insertional mutation in the
dnaK
-
dnaJ
operon of pathogenic strain χ3306. The DnaK/DnaJ-depleted mutant was temperature sensitive for growth, that is, nonviable above 39°C. We then isolated a spontaneously occurring revertant of the
dnaK
-
dnaJ
-disrupted mutant at 39°C and used it for infection of mice. The mutant lost the ability to cause a lethal systemic disease in mice. The impaired ability for virulence was restored when a functional copy of the
dnaK
-
dnaJ
operon was provided, suggesting that the DnaK/DnaJ chaperone machinery is required by
Salmonella
for the systemic infection of mice. This result also indicates that with respect to the DnaK/DnaJ chaperone machinery, the cellular requirements for growth at a high temperature are not identical to the cellular requirements for the pathogenesis of
Salmonella
. Macrophage survival assays revealed that the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages. Of further interest are the findings that the mutant could neither invade cultured epithelial cells nor secrete any of the invasion proteins encoded within
Salmonella
pathogenicity island 1. This is the first time that the DnaK/DnaJ chaperone machinery has been shown to be involved in bacterial invasion of epithelial cells.
Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in
Pseudomonas aeruginosa
Authors: Rietsch Arne , Wolfgang Matthew C. , Mekalanos John J. 
Published: February 2004

ABSTRACT

The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In
Pseudomonas aeruginosa
and
Yersinia
spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of
P. aeruginosa
PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in
exoS
expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in
cbrA
that also causes overexpression of
cbrB
. The
cbrAB
two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.
An NADPH Quinone Reductase of
Helicobacter pylori
Plays an Important Role in Oxidative Stress Resistance and Host Colonization
Authors: Wang Ge , Maier Robert J. 
Published: February 2004

ABSTRACT

Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the toxic reactive oxygen species released by the host. In a previous study characterizing oxidative stress resistance mutants of
Helicobacter pylori
, a novel potential antioxidant protein (MdaB) was identified by the observation that the expression of this protein was significantly upregulated to compensate for the loss of other major antioxidant components. In this study, we characterized an
H. pylori mdaB
mutant and the MdaB protein. While the wild-type strain can tolerate 10% oxygen for growth, the growth of the
mdaB
mutant was significantly inhibited by this oxygen condition. The
mdaB
mutant is also more sensitive to H
2
O
2
, organic hydroperoxides, and the superoxide-generating agent paraquat. Although the wild-type strain can survive more than 10 h of air exposure, exposure of the mutant strain to air for 8 h resulted in recovery of no viable cells. The oxidative stress sensitivity of the
mdaB
mutant resulted in a deficiency in the ability to colonize mouse stomachs.
H. pylori
was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with about 5,000 to 45,000 CFU/g of stomach. However, only 3 of 12 mice that were inoculated with the
mdaB
mutant strain were found to harbor any
H. pylori
, and these 3 contained less than 2,000 CFU/g of stomach. A His-tagged MdaB protein was purified and characterized. It was shown to be a flavoprotein that catalyzes two-electron transfer from NAD(P)H to quinones. It reduces both ubiquinones and menaquinones with similar efficiencies and preferably uses NADPH as an electron donor. We propose that the physiological function of the
H. pylori
MdaB protein is that of an NADPH quinone reductase that plays an important role in managing oxidative stress and contributes to successful colonization of the host.
Effects of RelA on Key Virulence Properties of Planktonic and Biofilm Populations ofStreptococcus mutans
Authors: Lemos José A. C. , Brown Thomas A. , Burne Robert A. 
Published: February 2004

ABSTRACTStreptococcus mutansis a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of theS. mutans relAgene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in whichrelAwas insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent andrelAstrains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of theluxSgene was increased as much as fivefold in therelAmutants, suggesting a link between AI-2 quorum sensing and the stringent response.
Invasion of Porcine Brain Microvascular Endothelial Cells by
Streptococcus suis
Serotype 2
Authors: Vanier Ghyslaine , Segura Mariela , Friedl Peter , Lacouture Sonia , Gottschalk Marcelo 
Published: February 2004

ABSTRACT

Streptococcus suis
is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of
S. suis
infection has not been completely defined. However, in order to cause meningitis,
S. suis
has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of
S. suis
serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European
S. suis
serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of
S. suis
seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable
S. suis
could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by
S. suis
required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by
S. suis
.
Paneth Cell α-Defensins from Rhesus Macaque Small Intestine
Authors: Tanabe Hiroki , Yuan Jun , Zaragoza Melinda M. , Dandekar Satya , Henschen-Edman Agnes , Selsted Michael E. , Ouellette Andre J. 
Published: February 2004

ABSTRACTAntimicrobial peptides are secreted by small intestinal Paneth cells as components of innate immunity. To investigate the role of α-defensins in enteric host defenses in nonhuman primates, α-defensin cDNAs were isolated, α-defensin peptides were purified from rhesus macaque small bowel, and the bactericidal activities of the peptides were measured. Six rhesus enteric α-defensin (RED) cDNAs, RED-1 to RED-6, were identified in a jejunum cDNA library; the deduced RED peptides exhibited extensive diversity relative to the primary structures of rhesus myeloid α-defensins. RED-4 was purified from monkey jejunum, and N-terminal peptide sequencing of putative RED-4 peptides identified two N termini, RTCYCRTGR… and TCYCRTGRC…; these corresponded to alternative N termini for the RED-4 molecules, as deduced from their molecular masses and RED cDNAs. In situ hybridization experiments localized RED mRNAs exclusively to small intestinal Paneth cells. Recombinant RED-1 to RED-4 were purified to homogeneity and shown to be microbicidal in the low micromolar range (≤10 μg/ml) against gram-positive and gram-negative bacteria, with individual peptides exhibiting variable target cell specificities. Thus, compared to myeloid α-defensins from rhesus macaques, enteric α-defensin peptides are highly variable in both primary structure and activity. These studies should facilitate further analyses of the role of α-defensins in primate enteric immunity.
γδ T Cells in Immunity Induced byMycobacterium bovisBacillus Calmette-Guérin Vaccination
Authors: Lee Jinhee , Choi Keumhwa , Olin Michael R. , Cho Sang-Nae , Molitor Thomas W. 
Published: February 2004

ABSTRACTMycobacterium bovisbacillus Calmette-Guérin (BCG) vaccination is efficacious for newborns or adults with no previous exposure to environmental mycobacteria. To determine the relative contribution and the nature of γδ T-cell receptor-positive T cells in newborns, compared to CD4+T cells, in immunity induced byM. bovisBCG vaccination, 4-week-old specific-pathogen-free pigs were vaccinated withM. bovisBCG and monitored by following the γδ T-cell immune responses. A flow cytometry-based proliferation assay and intracellular staining for gamma interferon (IFN-γ) were used to examine γδ T-cell responses. Pigs were found to mount Th1-like responses toM. bovisBCG vaccination as determined by immunoproliferation and IFN-γ production. The γδ T-cell lymphoproliferation and IFN-γ production to stimulation with mycobacterial antigens were significantly enhanced byM. bovisBCG vaccination. The relative number of proliferating γδ T cells after stimulating peripheral blood mononuclear cells withMycobacterium tuberculosisH37Rv culture filtrate protein was higher than that of CD4+T cells at an early time point afterM. bovisBCG vaccination, but CD4+T cells were found to be more abundant at a later time point. Although the γδ T-cell responses were dependent on the presence of CD4+T cells for the cytokine interleukin-2, the enhanced γδ T cells were due to the intrinsic changes of γδ T cells caused byM. bovisBCG vaccination rather than being due solely to help from CD4+T cells. Our study shows that γδ T cells from pigs at early ages are functionally enhanced byM. bovisBCG vaccination and suggests an important role for this T-cell subset in acquired immunity conferred byM. bovisBCG vaccination.
Increases in c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase and p38 Activity in Monocyte-Derived Macrophages following the Uptake of
Legionella pneumophila
Authors: Welsh Chad T. , Summersgill James T. , Miller Richard D. 
Published: February 2004

ABSTRACT

Legionella pneumophila
, the causative agent of Legionnaires' disease, infects and replicates within a variety of eukaryotic cells. The purpose of the current study was to examine host cell signaling events immediately following uptake and early in the endocytic process (less than 1 h) following the phagocytosis of
L. pneumophila
. This examination focused on the protein kinase signal pathways to identify any aberrant signal(s) induced by
L. pneumophila
within its host, as a means to alter the normal endocytic pathway. The mitogen-activated protein kinase cascades are of interest due to their involvement in cellular regulation. The experiments were carried out with monocyte-derived macrophages (MDMs). All three mitogen-activated protein kinase cascades were activated when MDMs were inoculated with either
Legionella
strain (wild-type strain AA100 or
dotA
mutant GL10) or an
Escherichia coli
control. Whereas the avirulent treatments, GL10 and
E. coli
, exhibited a leveling off or a return to near basal levels of phosphorylation/activity of c-Jun N-terminal kinase by 60 min, the virulent strain AA100 exhibited a significantly increased level of activity through 60 min that was greater than that seen in GL10 (
P
= 0.025) and
E. coli
(
P
= 0.014). A similar trend was seen with p38 phosphorylation. Phosphorylation of mitogen-activated protein/ERK kinase (MEK) was decreased in strain AA100 compared to
E. coli
. Inhibition of the activity of either the stress-activated protein kinase/c-Jun N-terminal kinase or p38 pathway significantly decreased the ability of legionellae to replicate intracellularly, suggesting the necessity of these two pathways in its intracellular survival and replication.
Populations of Human T Lymphocytes That Traverse the Vascular Endothelium Stimulated byBorrelia burgdorferiAre Enriched with Cells That Secrete Gamma Interferon
Authors: Gergel Edna I. , Furie Martha B. 
Published: February 2004

ABSTRACTSome diseases are characterized by prevalence in the affected tissues of type 1 T lymphocytes, which secrete gamma interferon (IFN-γ) and other proinflammatory cytokines. For example, type 1 T cells predominate in the lesions of patients with Lyme disease, which is caused by the bacteriumBorrelia burgdorferi. We used an in vitro model of the blood vessel wall to test the premise that the vascular endothelium actively recruits circulating type 1 T cells to such lesions. When T lymphocytes isolated from human peripheral blood were examined, the populations that traversed monolayers of resting human umbilical vein endothelial cells (HUVEC) or HUVEC stimulated by interleukin-1β orB. burgdorferiwere markedly enriched for T cells that produced IFN-γ compared to the initially added population of T cells. No enrichment was seen for cells that produced interleukin-4, a marker for type 2 T lymphocytes. Very late antigen-4 and CD11/CD18 integrins mediated passage of the T cells across both resting and stimulated HUVEC, and the endothelium-derived chemokine CCL2 (monocyte chemoattractant protein 1) was responsible for the enhanced migration of T cells across stimulated HUVEC. These results suggest that the vascular endothelium may contribute to the selective accumulation of type 1 T cells in certain pathological lesions, including those of Lyme disease.
TheBurkholderia cepaciaEpidemic Strain Marker Is Part of a Novel Genomic Island Encoding Both Virulence and Metabolism-Associated Genes inBurkholderia cenocepacia
Authors: Baldwin Adam , Sokol Pamela A. , Parkhill Julian , Mahenthiralingam Eshwar 
Published: February 2004

ABSTRACTTheBurkholderia cepaciaepidemic strain marker (BCESM) is a useful epidemiological marker for virulentB. cenocepaciastrains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): anN-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated theB. cenocepaciaisland (cci), is the first genomic island to be defined in theB. cepaciacomplex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.
Epitope Mapping of a Protective Monoclonal Antibody against
Pneumocystis carinii
with Shared Reactivity to
Streptococcus pneumoniae
Surface Antigen PspA
Authors: Wells Jesse , Gigliotti Francis , Simpson-Haidaris Patricia J. , Haidaris Constantine G. 
Published: February 2004

ABSTRACT

Pneumocystis carinii
is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived
P. carinii
was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of
P. carinii
derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse
P. carinii
antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse
P. carinii
recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen,
Streptococcus pneumoniae
PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the
S. pneumoniae
surface, and Western blotting identified a protein in
S. pneumoniae
lysates consistent with the

M
r

of PspA. A fragment of the
S. pneumoniae
PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in
P. carinii
. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize
P. carinii
derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against
P. carinii
.
Fine Specificity of Serum Antibodies toPlasmodium falciparumMerozoite Surface Protein, PfMSP-119, Predicts Protection from Malaria Infection and High-Density Parasitemia
Authors: Okech Brenda A. , Corran Patrick H. , Todd James , Joynson-Hicks Amy , Uthaipibull Chairat , Egwang Thomas G. , Holder Anthony A. , Riley Eleanor M. 
Published: February 2004

ABSTRACTAntibodies to the C terminus of thePlasmodium falciparummerozoite surface protein, PfMSP-119, may inhibit merozoite invasion or block the effects of inhibitory antibodies. Here, using a competition enzyme-linked immunosorbent assay and antibody binding to wild-type and mutated recombinant proteins, we show that there are marked variations between individuals in the fine specificity of naturally acquired anti-MSP-119antibodies. Furthermore, although neither the prevalence nor the concentration of total anti-MSP-119antibodies was associated with resistance to malaria in African children, significant associations were observed between antibody fine specificity and subsequent risk of infection and high-density parasitemia during a follow-up period. Thus, the fine specificity of naturally acquired human anti-MSP-119antibodies is crucial in determining their function. Future field studies, including the evaluation of PfMSP-1 vaccine trials, should include assays that explore antibody fine specificity as well as titer.
A Locus Contained within a Variable Region of Pneumococcal Pathogenicity Island 1 Contributes to Virulence in Mice
Authors: Brown Jeremy S. , Gilliland Sarah M. , Spratt Brian G. , Holden David W. 
Published: February 2004

ABSTRACT

We have previously described a 27-kb pathogenicity island of
Streptococcus pneumoniae
, termed pneumococcal pathogenicity island 1 (PPI1), which contains iron uptake locus
piaABCD
, required for full virulence in mice, and a further 28 previously uncharacterized genes. We have investigated one of these, Sp1051, which encodes a protein of unknown function. Disruption of Sp1051 does not affect growth in laboratory broth, serum, or blood but impairs virulence in mouse models of infection. When
S. pneumoniae
capsular serotypes were analyzed by PCR and Southern hybridization, it was found that 33% did not contain Sp1051. Analysis of other genes within PPI1 demonstrated that, compared to the serotype 4 genome published by The Institute for Genome Research (TIGR), the genomes of many strains contain deletions of a variable number of genes between Sp1046 and Sp1064, conforming to one of six different patterns. Amplification by PCR of this PPI1 variable region from a capsular serotype 17 strain and comparison of the sequence to TIGR serotype 4 strain sequence showed that Sp1051 is contained within an 11.3-kb segment of DNA flanked by 7-bp direct repeats within the serotype 4 strain which is not present in the serotype 17 strain. Further comparison of the sequences of this region between the three published
S. pneumoniae
genomes demonstrated that serotype 19F and strain R6 contain novel complements of genes not present in the serotype 4 strain. These data indicate that there is striking variation in gene content and structure of the 3′ region of PPI1 among strains and that this region includes at least one virulence determinant. Gene variation within horizontally acquired DNA such as that of PPI1 may be one factor modulating differences in virulence among strains.