bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

Live 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

Antibodies 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

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

BALB/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

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.
Antibody Avidity and Immunoglobulin G Isotype Distribution following Immunization with a Monovalent Meningococcal B Outer Membrane Vesicle Vaccine
Authors: Vermont C. L. , van Dijken H. H. , van Limpt C. J. P. , de Groot R. , van Alphen L. , van den Dobbelsteen G. P. J. M.
Published: July 2002

The avidity maturation and immunoglobulin G (IgG) isotype distribution of antibodies after vaccination with a meningococcal B outer membrane vesicle (OMV) vaccine were evaluated as indicators of protective immunity. Pre- and postvaccination sera from 134 healthy toddlers (ages, 2 to 3 years) immunized with a monovalent meningococcal B OMV (serosubtype P1.7-2,4) vaccine adsorbed with AlPO4or Al(OH)3were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. The children were vaccinated three times with intervals of 3 to 6 weeks between vaccinations or twice with an interval of 6 to 10 weeks between vaccinations. A booster was given after 20 to 40 weeks. The avidity index (AI) of antibodies increased significantly during the primary series of vaccinations and after the booster was given. No differences in AIs were found when the results obtained with the two vaccination schedules or with the two adjuvants were compared. After vaccination, IgG1 was the predominant IgG isotype, followed by IgG3. No IgG2 or IgG4 was detected. There was a strong correlation between serum bactericidal activity (SBA) and ELISA titers (r= 0.85 [P< 0.0001] for total IgG,r= 0.83 for IgG1 [P< 0.0001],r= 0.82 for IgG3 [P< 0.0001], andr= 0.84 [P< 0.0001] for the avidity titer). When two subgroups with similar anti-OMV IgG levels were compared before and after the booster vaccination, the higher AI after the booster vaccination was associated with significantly increased SBA. We concluded that avidity maturation occurs after vaccination with a monovalent meningococcal B OMV vaccine, especially after boosting, as indicated by a significant increase in the AI. Vaccination with the monovalent OMV vaccine induced mainly IgG1 and IgG3 isotypes, which are considered to be most important for protection against meningococcal disease. An increase in the AI of antibodies is associated with increased SBA, independent of the level of specific IgG and the IgG isotype distribution. Measuring the AI and IgG isotype distribution of antibodies after vaccination can be a supplementary method for predicting protective immunity for evaluation in future phase III trials with meningococcal serogroup B vaccines.
Dominant-Negative Rho, Rac, and Cdc42 Facilitate the Invasion Process of
Vibrio parahaemolyticus
into Caco-2 Cells
Authors: Akeda Yukihiro , Kodama Toshio , Kashimoto Takashige , Cantarelli Vlademir , Horiguchi Yasuhiko , Nagayama Kenichi , Iida Tetsuya , Honda Takeshi
Published: July 2002

To clarify the invasive process of
Vibrio parahaemolyticus
, an invasion assay was performed using cells expressing dominant negative small GTPases of the Rho family. This assay showed that the dominant negative host phenotype facilitates bacterial invasion, suggesting that the mechanism of
V. parahaemolyticus
invasion differs from that reported for other invasive bacteria.
HLA-B*35-Restricted CD8+-T-Cell Epitope inMycobacterium tuberculosisRv2903c
Authors: Klein Michèl R. , Hammond Abdulrahman S. , Smith Steve M. , Jaye Assan , Lukey Pauline T. , McAdam Keith P. W. J.
Published: July 2002

Few human CD8+T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8+T-cell epitope inMycobacterium tuberculosisRv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to killM. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.
Correlation between an In Vitro Invasion Assay and a Murine Model ofBurkholderia cepaciaLung Infection
Authors: Cieri Martin V. , Mayer-Hamblett Nicole , Griffith Adam , Burns Jane L.
Published: July 2002

Our understanding of the virulence ofBurkholderia cepaciacomplex lung infections in cystic fibrosis patients is incomplete. There is a great deal of variability in the clinical course, from simple colonization to severe and often fatal necrotizing pneumonia, termed cepacia syndrome. Multiple subspecies (called genomovars) have been identified, and these genomovars may hold the key to understanding the variable pathogenicity. Thirty-oneB. cepaciacomplex isolates belonging to five of the seven genomovars were examined by using a gentamicin protection assay of invasion with A549 cells. The level of epithelial cell invasion byB. cepaciain the A549 model was relatively low compared with the data obtained for other pathogens and was often variable from assay to assay. Thus, a statistical approach was used to determine invasiveness. When this model was used, one of four genomovar I strains (25%), three of eight genomovar II strains (37.5%), seven of nine genomovar III strains (77.8%), one of four genomovar IV strains (25%), and none of the four genomovar V strains examined were defined as invasive. All other strains were categorized as either noninvasive or indeterminate. Invasive, noninvasive, and indeterminate isolates belonging to genomovars II and III were subsequently tested for splenic invasion with the mouse agar bead model. Correlation between the models for six strains was demonstrated. Our results indicate that a statistical model used to determine invasiveness in an in vitro invasion assay can be used to predict in vivo invasiveness.
Genetic Variability and Stability of
Anaplasma phagocytophila msp2
(
p44
)
Authors: Caspersen Karen , Park Jin-Ho , Patil Surekha , Dumler J. Stephen
Published: July 2002

Anaplasma
(
Ehrlichia
)
phagocytophila
's major immunodominant surface protein antigen, Msp2 (P44, 44-kDa antigen), is encoded by a family of paralogous genes characterized by conserved sequences flanking a hypervariable region. The antigenic profiles of most strains of
A. phagocytophila
are different, and the differences are principally related to Msp2 expression. To date, multiple unique
msp2
gene paralogs have been found in
A. phagocytophila
isolates, but the overall number in the genome of a single strain is not yet known. Changes in
msp2
expression may be related to antigenic variability; thus, we examined the minimal complement of
msp2
genes or pseudogenes in two strains of
A. phagocytophila
and the number of transcriptionally active
msp2
gene paralogs during low-passage, steady-state, in vitro propagation. Of 15 BDS strain clones, 1 had a hypervariable region identical to the region in a clone obtained from a BDS strain genomic library previously prepared from organisms after only two horse passages. When 124 Webster strain clones were examined, 18 unique hypervariable regions were identified. Of 64 Webster strain cDNA clones, 56 (87.5%) were derived from a single gene, and transcripts from six additional
msp2
genes were also identified. The sequences of several hypervariable regions that were ≥97% similar to regions present in other strains were identified by performing a BLAST analysis of sequences deposited in the GenBank database. These findings suggest that antigenic variability results from transcription of one or a few of the multiple paralogs and not from genetic instability that results in random accumulated mutations, although the possibility that gene recombination plays a role cannot be eliminated. The predominant Msp2 pattern in vitro is determined by transcription from a single gene.
Effect ofMycobacterium bovisBCG Vaccination on Interleukin-1β and RANTES mRNA Expression in Guinea Pig Cells Exposed to Attenuated and Virulent Mycobacteria
Authors: Jeevan Amminikutty , Yoshimura Teizo , Foster Gregory , McMurray David N.
Published: July 2002

The effect ofMycobacterium bovisBCG vaccination on interleukin-1β (IL-1β) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected withM. bovisBCG,Mycobacterium avium, the attenuatedMycobacterium tuberculosisH37Ra strain, or virulent strains H37Rv and Erdman ofM. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1β or RANTES cDNA. Although IL-1β and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain ofM. tuberculosisshowed 75 to 90% reductions in IL-1β expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1β mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h afterM. tuberculosisH37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulentM. tuberculosis.These initial studies indicate that BCG vaccination has a positive effect on IL-1β and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.
The Sortase SrtA of
Listeria monocytogenes
Is Involved in Processing of Internalin and in Virulence
Authors: Garandeau Caroline , Réglier-Poupet Hélène , Dubail Iharilalao , Beretti Jean-Luc , Berche Patrick , Charbit Alain
Published: July 2002

Listeria monocytogenes
is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in
Staphylococcus aureus
that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of
L. monocytogenes
a gene, designated
srtA
, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the
srtA
mutation on cell wall anchoring of this LPXTG protein in
L. monocytogenes
. We show that the
srtA
mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that
srtA
of
L. monocytogenes
acts as a sortase and plays a role in the pathogenicity.
Interaction of an Outer Membrane Protein of Enterotoxigenic
Escherichia coli
with Cell Surface Heparan Sulfate Proteoglycans
Authors: Fleckenstein James M. , Holland James T. , Hasty David L.
Published: July 2002

We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic
Escherichia coli
(ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant
E. coli
expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant
E. coli
expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.
Influence of Recombination and Niche Separation on the Population Genetic Structure of the Pathogen
Streptococcus pyogenes
Authors: Kalia Awdhesh , Spratt Brian G. , Enright Mark C. , Bessen Debra E.
Published: July 2002

The throat and skin of the human host are the principal reservoirs for the bacterial pathogen
Streptococcus pyogenes
. The
emm
locus encodes structurally heterogeneous surface fibrils that play numerous roles in virulence, depending on the strain. Isolates harboring the
emm
pattern A-C marker exhibit a strong tendency to cause throat infection, whereas
emm
pattern D strains are usually recovered from impetigo lesions; as a group,
emm
pattern E organisms fail to display obvious tissue tropisms. The peak incidence for streptococcal pharyngitis and impetigo varies with season and locale, leading to wide spatial and temporal distances between throat and skin strains. To assess any impact of niche separation on genetic variation, the extent of recombinational exchange between
emm
pattern A-C, D, and E subpopulations was evaluated. Analysis of nucleotide sequence data for internal portions of seven housekeeping loci from 212 isolates provides evidence of extensive recombination between strains belonging to different
emm
pattern subpopulations. Furthermore, no fixed nucleotide differences were found between
emm
pattern A-C and D strains. Thus, despite some niche separation created by distinct epidemiological trends and innate tissue tropisms there is little evidence for neutral gene divergence between throat and skin strains. Maintenance of a relationship between
emm
pattern and tissue tropism in the face of underlying recombination suggests that tissue tropism is associated with
emm
or a closely linked gene.
Vaccination of Guinea Pigs with DNA Encoding the Mycobacterial Antigen MPB83 Influences Pulmonary Pathology but Not Hematogenous Spread following Aerogenic Infection withMycobacterium bovis
Authors: Chambers Mark A. , Williams Ann , Hatch Graham , Gavier-Widén Dolores , Hall Graham , Huygen Kris , Lowrie Douglas , Marsh Philip D. , Hewinson R. Glyn
Published: July 2002

Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistentMycobacterium bovisinfection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from whichM. boviscould be excreted. Recent reports of successful DNA vaccination againstMycobacterium tuberculosisin small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolizedM. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembledM. bovisBCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection withM. bovis.
Role ofNeisseria meningitidis luxSin Cell-to-Cell Signaling and Bacteremic Infection
Authors: Winzer Klaus , Sun Yao-hui , Green Andrew , Delory Marie , Blackley David , Hardie Kim R. , Baldwin Thomas J. , Tang Christoph M.
Published: July 2002

Numerous pathogenic bacteria containluxS, which is required for autoinducer-2 production. Here, we demonstrate thatNeisseria meningitidiscontains a functional copy ofluxSthat is necessary for full meningococcal virulence; strains with aluxSdeletion are defective for bacteremia, a prerequisite of meningococcal pathogenesis.
Pirfenidone Blocks the In Vitro and In Vivo Effects of Staphylococcal Enterotoxin B
Authors: Hale Martha L. , Margolin Solomon B. , Krakauer Teresa , Roy Chad J. , Stiles Bradley G.
Published: July 2002

Pirfenidone [5-methyl-1-phenyl-2-(1H)-pyridone] down-regulates expression of cytokines and other mediators involved in the onset and development of pulmonary fibrosis. Pirfenidone also inhibits production of tumor necrosis factor alpha (TNF-α) from macrophages incubated with endotoxin and protects mice against endotoxin shock. Pirfenidone's ability to reduce cytokine expression in these disorders led us to investigate the drug's effect on another cytokine anomaly, superantigen-induced shock. BALB/c mice were exposed to staphylococcal enterotoxin B (SEB) either systemically or by aerosol and subsequently potentiated with a sublethal dose of lipopolysaccharide. In these experiments, pirfenidone given 2 to 4.25 h after SEB resulted in 80 to 100% survival versus only 0 to 10% survival among untreated control animals. Relative to serum cytokine levels from controls given toxin but no drug, there was a 35 to 80% decrease in TNF-α, interleukin 1, and other proinflammatory cytokines. In vitro experiments with human peripheral blood lymphocytes revealed that pirfenidone reduced SEB-induced cytokine levels 50 to 80% and inhibited 95% of SEB-induced T-cell proliferation. Overall, these studies demonstrated the potential utility of pirfenidone as a therapeutic against septic shock and the biological effects of SEB.
Quorum-Sensing
Escherichia coli
Regulator A: a Regulator of the LysR Family Involved in the Regulation of the Locus of Enterocyte Effacement Pathogenicity Island in Enterohemorrhagic
E. coli
Authors: Sperandio Vanessa , Li Caiyi C. , Kaper James B.
Published: July 2002

The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic
Escherichia coli
(EHEC) and enteropathogenic
E. coli
(EPEC). The LEE comprises 41 open reading frames organized in five major operons,
LEE1
,
LEE2
,
LEE3
,
tir
(
LEE5
), and
LEE4
, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of
LEE1
encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the
E. coli
genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and
E. coli
K-12; and shares homology with the AphB and PtxR regulators of
Vibrio cholerae
and
Pseudomonas aeruginosa
, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing
E. coli
regulator A (QseA). We observed that QseA activated transcription of
ler
and therefore of the other LEE genes. An EHEC
qseA
mutant had a striking reduction of type III secretion activity, which was complemented when
qseA
was provided in
trans
. Similar results were also observed with a
qseA
mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.
Identification ofChlamydia pneumoniae-Derived Mouse CD8 Epitopes
Authors: Sarén Anne , Pascolo Steve , Stevanovic Stefan , Dumrese Tilman , Puolakkainen Mirja , Sarvas Matti , Rammensee Hans-Georg , Vuola Jenni M.
Published: July 2002

Chlamydia pneumoniaeis a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine againstC. pneumoniaeinfection, but CD8+T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8+T cell remain unknown. The aim of this study was to identifyC. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of fourC. pneumoniaeproteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8+T-cell epitopes were predicted byH-2class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented byC. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition ofC. pneumoniae-infected target cells. By using this strategy, we were able to identify threeC. pneumoniaeCD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8+T-cell function in vivo and generation of epitope-specific prevention strategies.
Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation inLeishmania amazonensisAmastigotes
Authors: Holzmuller Philippe , Sereno Denis , Cavaleyra Mireille , Mangot Isabelle , Daulouede Sylvie , Vincendeau Philippe , Lemesre Jean-Loup
Published: July 2002

Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through thel-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoanLeishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO2or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes ofL. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation inLeishmaniaamastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets.
Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with
Corynebacterium glutamicum
during Growth on Acetate and/or Glucose
Authors: Wendisch Volker F. , de Graaf Albert A. , Sahm Hermann , Eikmanns Bernhard J.
Published: July 2002

Growth of
Corynebacterium glutamicum
on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By
13
C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of
C. glutamicum
were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol · (mg of protein)
−1
· min
−1
, respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol · [mg of protein]
−1
· min
−1
) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol · [mg of protein]
−1
· min
−1
). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol · [mg of protein]
−1
· min
−1
) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of
C. glutamicum
, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.
Structural Elements Required for Replication and Incompatibility of the
Rhizobium etli
Symbiotic Plasmid
Authors: Ramírez-Romero Miguel A. , Soberón Nora , Pérez-Oseguera Angeles , Téllez-Sosa Juan , Cevallos Miguel A.
Published: July 2002

The symbiotic plasmid of
Rhizobium etli
CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes,
repA
,
repB
, and
repC
, encoded by the same DNA strand. A long intergenic sequence (
igs
) between
repB
and
repC
is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the
repABC
genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two
cis
-acting incompatibility regions, one located in the
igs
(
inc
α) and the other downstream of
repC
(
inc
β) (the former is essential for replication); and (v) RepA is a
trans
-acting incompatibility factor. We suggest that
inc
α is a
cis
-acting site required for plasmid partitioning and that the origin of replication lies within
inc
β.
Integration and Excision of a
Bacteroides
Conjugative Transposon, CTnDOT
Authors: Cheng Qi , Paszkiet Brian J. , Shoemaker Nadja B. , Gardner Jeffrey F. , Salyers Abigail A.
Published: July 2002

Bacteroides
conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most
Bacteroides
CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the
Bacteroides
CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF),
intDOT
, was necessary and sufficient for integration into the
Bacteroides
chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn
916
, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing
intDOT
and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of
intDOT
designated
orf2
. Thus, excision involves other genes besides
intDOT
and
orf2
. Both
intDOT
and
orf2
were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that
intDOT
was essential for excision but
orf2
was not. Despite its proximity to the integrase gene,
orf2
appears not to be essential for excision.
3-Deoxy-
d
-
manno
-oct-2-ulosonic Acid (Kdo) Transferase of
Legionella pneumophila
Transfers Two Kdo Residues to a Structurally Different Lipid A Precursor of
Escherichia coli
Authors: Brabetz Werner , Schirmer Christiane E. , Brade Helmut
Published: July 2002

The 3-deoxy-
d
-
manno
-oct-2-ulosonic acid (Kdo) transferase gene of
Legionella pneumophila
was cloned and sequenced. Despite remarkable structural differences in lipid A, the gene complemented a corresponding
Escherichia coli
mutant and was shown to encode a bifunctional enzyme which transferred 2 Kdo residues to a lipid A acceptor of
E. coli.
Regulation of Stalk Elongation by Phosphate in
Caulobacter crescentus
Authors: Gonin Madeleine , Quardokus Ellen M. , O'Donnol Danielle , Maddock Janine , Brun Yves V.
Published: July 2002

In
Caulobacter crescentus
, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes
pstSCAB
of
Escherichia coli
. In
E. coli
,
pst
mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the
Caulobacter phoB
gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While
phoB
was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both
pstS
and
phoB
mutants were deficient in phosphate transport. When a
phoB
mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of
phoB
and
pst
mutants were opposite.
phoB
mutants were unable to elongate stalks during phosphate starvation, whereas
pst
mutants made long stalks in both high- and low-phosphate media. Analysis of double
pst phoB
mutants indicated that the long-stalk phenotype of
pst
mutants was dependent on
phoB
. In addition, analysis of a
pstS-lacZ
transcriptional fusion showed that
pstS
transcription is dependent on
phoB
. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.
Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin
Authors: Gründling Angelika , Bläsi Udo , Young Ry
Published: July 2002

Bacteriophage λ uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. λ S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from λ S
C51S
were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S
C51S
(i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S
A52V
mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S
A52V/C51S
is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S
A52V
. Both S
A52V
(dominant negative) and S
A48V
(antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S
A52V
and S
A48V
, respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.
Metabolism of Acyl-Homoserine Lactone Quorum-Sensing Signals by
Variovorax paradoxus
Authors: Leadbetter Jared R. , Greenberg E. P.
Published: July 2002

Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class
Proteobacteria
. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize
N
-(3-oxohexanoyl)-
l
-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of
Variovorax paradoxus
. The
V. paradoxus
isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of
V. paradoxus
degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by
V. paradoxus
.
Correction of the DNA Sequence of theregBGene ofRhodobacter capsulatuswith Implications for the Membrane Topology of the Sensor Kinase RegB
Authors: Chen Weizhen , Jäger Andreas , Klug Gabriele
Published: July 2002

We corrected the previously published sequence for theregBgene, which encodes a histidine sensor kinase inRhodobacter capsulatus. The deduced RegB amino acid sequence has an additional putative transmembrane domain at the N terminus. Analysis of RegB-PhoA and RegB-LacZ fusion proteins supports a topology model for RegB with six membrane-spanning domains.