bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pathoadaptive Mutations That Enhance Virulence: Genetic Organization of the
cadA
Regions of
Shigella
spp
Authors: Day William A. , Fernández Reinaldo E. , Maurelli Anthony T. 
Published: July 2002

ABSTRACT

Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in
Shigella
species that have evolved from LDC-expressing
Escherichia coli
. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of
Shigella
enterotoxins and that the gene encoding LDC,
cadA
, was abolished by large chromosomal deletions in each
Shigella
species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of the
cad
region were sequenced from the four
Shigella
species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral
E. coli
chromosome displaced the
cadA
locus to form distinct genetic linkages that are unique to each
Shigella
species. Hybridization studies, using an
E. coli
K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the
Shigella
genomes. None of these novel gene arrangements were observed in representatives of all
E. coli
phylogenies. Collectively, these observations indicate that inactivation of the
cadA
antivirulence gene occurred independently in each
Shigella
species. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal
E. coli
, strong pressures in host tissues selected
Shigella
clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.
Synergistic Effect of Muramyldipeptide with Lipopolysaccharide or Lipoteichoic Acid To Induce Inflammatory Cytokines in Human Monocytic Cells in Culture
Authors: Yang Shuhua , Tamai Riyoko , Akashi Sachiko , Takeuchi Osamu , Akira Shizuo , Sugawara Shunji , Takada Haruhiko 
Published: July 2002

ABSTRACTAn analog of 1α,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects withSalmonellalipopolysaccharide (LPS) or lipoteichoic acid (LTA) fromStaphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.
Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6
Authors: Olsen Anja Weinreich , van Pinxteren Laurens A. H. , Okkels Limei Meng , Rasmussen Peter Birk , Andersen Peter 
Published: July 2002

ABSTRACTIn this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level ofMycobacterium bovisBCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.
Distribution and Kinetics of Lipoprotein-Bound Endotoxin
Authors: Levels J. H. M. , Abraham P. R. , van den Ende A. , van Deventer S. J. H. 
Published: July 2002

ABSTRACTLipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (±8%) high-density lipoprotein (HDL), 25% (±7%) low-density lipoprotein, and 12% (±5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 μg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.
Molecular Basis for Immunoglobulin M Specificity to Epitopes inCryptococcus neoformansPolysaccharide That Elicit Protective and Nonprotective Antibodies
Authors: Nakouzi Antonio , Valadon Philippe , Nosanchuk Joshua , Green Nancy , Casadevall Arturo 
Published: July 2002

ABSTRACTThe protective efficacy of antibodies (Abs) toCryptococcus neoformansglucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype DC. neoformans,while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (VH) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 VHbinding cavity to the corresponding residues in the 13F1 VHaltered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy againstC. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.
PspC, a Pneumococcal Surface Protein, Binds Human Factor H
Authors: Dave Sandhya , Brooks-Walter Alexis , Pangburn Michael K. , McDaniel Larry S. 
Published: July 2002

ABSTRACT

PspC was found to bind human complement factor H (FH) by Western blot analysis of D39 (
pspC
+
) and an isogenic mutant TRE108 (
pspC
). We confirmed that PspA does not bind FH, while purified PspC binds FH very strongly. The binding of FH to exponentially growing pneumococci varied among different isolates when analyzed by fluorescence activated cell sorting analysis.
Normal Human Fibroblasts Express Pattern Recognition Receptors for Fungal (1→3)-β-d-Glucans
Authors: Kougias Panagiotis , Wei Duo , Rice Peter J. , Ensley Harry E. , Kalbfleisch John , Williams David L. , Browder I. William 
Published: July 2002

ABSTRACTFungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (KD) of 8.9 ± 1.9 μg of protein per ml and a maximum binding of 100 ± 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with aKDof 5.6 μM (95% confidence interval [CI], 3.0 to 11 μM), while laminarin competed for 69% ± 6% of binding sites, with aKDof 3.7 μM (95% CI, 1.9 to 7.3 μM). Glucan (1 μg/ml) stimulated fibroblast NF-κB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-κB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1→3)-β-d-glucans and suggests that the response to glucans may not be confined to cells of the immune system.
Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of
Mycobacterium tuberculosis
Authors: Dahl John L. , Wei Jun , Moulder James W. , Laal Suman , Friedman Richard L. 
Published: July 2002

ABSTRACT

Mycobacterium tuberculosis
is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how
M. tuberculosis
avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in
M. tuberculosis
, designated
eis
, was found to enhance intracellular survival of
Mycobacterium smegmatis
in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). When
eis
was introduced into
M. smegmatis
on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the
eis
gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of
eis
. The Eis protein produced by
M. tuberculosis
H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced
eis
protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of
M. tuberculosis
H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of
M. tuberculosis
by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important
M. tuberculosis
immunogen.
In Vivo Visualization of Bacterial Colonization, Antigen Expression, and Specific T-Cell Induction following Oral Administration of Live RecombinantSalmonella entericaSerovar Typhimurium
Authors: Bumann Dirk 
Published: July 2002

ABSTRACTLive attenuatedSalmonellastrains that express a foreign antigen are promising oral vaccine candidates. Numerous genetic modifications have been empirically tested, but their effects on immunogenicity are difficult to interpret since important in vivo properties of recombinantSalmonellastrains such as antigen expression and localization are incompletely characterized and the crucial early inductive events of an immune response to the foreign antigen are not fully understood. Here, methods were developed to directly localize and quantitate the in situ expression of an ovalbumin model antigen in recombinantSalmonella entericaserovar Typhimurium using two-color flow cytometry and confocal microscopy. In parallel, the in vivo activation, blast formation, and division of ovalbumin-specific CD4+T cells were followed using a well-characterized transgenic T-cell receptor mouse model. This combined approach revealed a biphasic induction of ovalbumin-specific T cells in the Peyer's patches that followed the local ovalbumin expression of orally administered recombinantSalmonellacells in a dose- and time-dependent manner. Interestingly, intactSalmonellacells and cognate T cells seemed to remain in separate tissue compartments throughout induction, suggesting a transport of killedSalmonellacells from the colonized subepithelial dome area to the interfollicular inductive sites. The findings of this study will help to rationally optimize recombinantSalmonellastrains as efficacious live antigen carriers for oral vaccination.
Selection of Glutamate-Rich Protein Long Synthetic Peptides for Vaccine Development: Antigenicity and Relationship with Clinical Protection and Immunogenicity
Authors: Theisen Michael , Dodoo Daniel , Toure-Balde Aissatou , Soe Soe , Corradin Giampietro , Koram Kwadwo K. , Kurtzhals Jørgen A. L. , Hviid Lars , Theander Thor , Akanmori Bartholomew , Ndiaye Mohamadou , Druilhe Pierre 
Published: July 2002

ABSTRACTAntibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) ofPlasmodium falciparumwere analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinicalP. falciparummalaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of differentH-2haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination againstP. falciparummalaria.
The Global Regulator ArcA Controls Resistance to Reactive Nitrogen and Oxygen Intermediates in
Salmonella enterica
Serovar Enteritidis
Authors: Lu Sangwei , Killoran Patrick B. , Fang Ferric C. , Riley Lee W. 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Enteritidis is a major cause of food-borne diseases associated with consumption of shell eggs. Clinical isolates of
S. enterica
serovar Enteritidis exhibit a wide spectrum of virulence in mice. A highly virulent isolate (SE2472) was previously shown to be more resistant in vitro than other clinical isolates to acidified sodium nitrite (ASN), a generator of reactive nitrogen and oxygen intermediates (RNI/ROI). SE2472 is also more resistant to
S
-nitrosoglutathione (GSNO) and hydrogen peroxide (H
2
O
2
) than an ASN-susceptible isolate of
S. enterica
serovar Enteritidis (SE8743). To investigate the molecular basis for the RNI/ROI resistance of
S. enterica
serovar Enteritidis, we transformed a genomic DNA library of SE2472 into SE8743. A plasmid clone conferred upon SE8743 enhanced resistance to ASN, GSNO, and H
2
O
2
. The DNA insert in the clone encoded ArcA, a global regulator. An
arcA
mutant of SE2472 was constructed and was found to be more susceptible to GSNO and hydrogen peroxide but not more susceptible to ASN than wild-type SE2472. The susceptibility of the
arcA
mutant to GSNO and H
2
O
2
was complemented by a plasmid harboring
arcA
. The coding sequence of the
arcA
gene in SE2472 and the coding sequence of the
arcA
gene in SE8743 were identical, suggesting that the difference in resistance to RNI/ROI maybe due to the activity of genes regulated by ArcA. No significant difference in virulence between the wild type and the
arcA
mutant of SE2472 was observed in mice. These observations show that
arcA
is essential for resistance of
S. enterica
serovar Enteritidis to nitrosative and oxidative stress. However, additional genetic loci may contribute to the resistance to RNI/ROI and unusually high virulence for mice of SE2472.
Influence of Parasite Load on the Ability of Type 1 T Cells To ControlLeishmania majorInfection
Authors: Hondowicz Brian , Scott Phillip 
Published: July 2002

ABSTRACTBALB/c mice infected withLeishmania majordeveloped a type 2 immune response which failed to control parasite replication. We found thatscidmice that received splenocytes from BALB/c mice that had been infected for 3 weeks withL. major(a type 2 cell population) and that were subsequently infected withL. majorwere protected when they were treated with interleukin 12 (IL-12). In contrast, IL-12 was ineffective at protecting BALB/c mice infected for 3 weeks, suggesting that a high parasite load regulates the development of protective immunity. To determine how this regulation operates, we performed a series of adoptive transfers of naïve, type 1 or type 2 splenocytes intoscidmice. The recipientscidmice were infected either for 5 weeks prior to cell transfer (and thus had a high parasite load) or at the time of cell transfer.scidmice that were infected for 5 weeks and received a type 1 cell population were able to cure their lesions. However, when 5-week-infectedscidmice received both type 1 and 2 cell populations, they were unable to control their infections. In contrast, the same type 1 and 2 cells transferred to naïvescidmice, which were subsequently infected, provided protection. In addition, we found that naïve cells mediated protection inscidmice with established lesions. These results show that high parasite numbers do not block type 1 protective responses or the development of type 1 responses. Instead, the influence of a high parasite load is dependent on the presence of a type 2 cell population.
Efficiency of Protection of Guinea Pigs against Infection with
Bacillus anthracis
Spores by Passive Immunization
Authors: Kobiler David , Gozes Yehoshua , Rosenberg Hagai , Marcus Dino , Reuveny Shaul , Altboum Zeev 
Published: July 2002

ABSTRACT

The efficacy of passive immunization as a postexposure prophylactic measure for treatment of guinea pigs intranasally infected with
Bacillus anthracis
spores was evaluated. Antisera directed either against the lethal toxin components (PA or LF) or against a toxinogenic strain (Sterne) were used for this evaluation. All antisera exhibited high enzyme-linked immunosorbent assay titers against the corresponding antigens, high titers of neutralization of cytotoxicity activity in an in vitro mouse macrophages cell line (J774A.1), as well as in vivo neutralization of toxicity when administered either directly to Fisher rats prior to challenge with the lethal toxin or after incubation with the lethal toxin. In these tests, anti-LF antiserum exhibited the highest neutralization efficiency, followed by anti-Sterne and anti-PA. The time dependence and antibody dose necessary for conferring postexposure protection by the various antibodies of guinea pigs infected with 25 50% lethal doses of Vollum spores was examined. Rabbit anti-PA serum was found to be the most effective. Intraperitoneal injections of anti-PA serum given 24 h postinfection protected 90% of the infected animals, whereas anti-Sterne and anti-LF were less effective. These results further emphasizes the importance of anti-PA antibodies in conferring protection against
B. anthracis
infection and demonstrated the ability of such antibodies to be effectively applied as an efficient postexposure treatment against anthrax disease.