bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunization with a Polyprotein Vaccine Consisting of the T-Cell Antigens Thiol-Specific Antioxidant,Leishmania majorStress-Inducible Protein 1, andLeishmaniaElongation Initiation Factor Protects against Leishmaniasis
Authors: Coler Rhea N. , Skeiky Yasir A. W. , Bernards Karen , Greeson Kay , Carter Darrick , Cornellison Charisa D. , Modabber Farrokh , Campos-Neto Antonio , Reed Steven G. 
Published: July 2002

ABSTRACTDevelopment of an effective vaccine againstLeishmaniainfection is a priority of tropical disease research. We have recently demonstrated protection againstLeishmania majorin the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant,L. majorstress-inducible protein 1 (LmSTI1), andLeishmaniaelongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using theL. majorchallenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.
Effect of Acid Adaptation on the Fate of
Listeria monocytogenes
in THP-1 Human Macrophages Activated by Gamma Interferon
Authors: Conte Maria Pia , Petrone Gloria , Di Biase Assunta Maria , Longhi Catia , Penta Michela , Tinari Antonella , Superti Fabiana , Fabozzi Giulia , Visca Paolo , Seganti Lucilla 
Published: July 2002

ABSTRACT

In
Listeria monocytogenes
the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of
L. monocytogenes
in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of
L. monocytogenes
in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the
plcA
gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic
plcA-prfA
transcript, concomitant with an increase in the level(s) of the monocistronic
prfA
mRNA(s). The transcriptional shift from distal to proximal
prfA
promoters resulted in equal levels of the
prfA
transcript (and, as a consequence, of the
inlA
,
hly
, and
actA
transcripts) under neutral and acidic conditions. In contrast, the
sodC
and
gad
genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted
L. monocytogenes
cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of
L. monocytogenes
with macrophagic cells.
Molecular Cloning and Characterization of Genes for
Shigella sonnei
Form I O Polysaccharide: Proposed Biosynthetic Pathway and Stable Expression in a Live
Salmonella
Vaccine Vector
Authors: Xu De-Qi , Cisar John O. , Ambulos Nicholas , Burr Donald H. , Kopecko Dennis J. 
Published: July 2002

ABSTRACT

The gene region for biosynthesis of
Shigella sonnei
form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of
Salmonella
-based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS
630
. A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS
91
and IS
630
) and
wzz
upstream of the form I coding region and a fragment of
aqpZ
and additional full or partial IS elements (i.e., IS
629
, IS
91
, and IS
911
) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS
91
from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated
Shigella
and
Escherichia coli
extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated
Salmonella enterica
serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent
S. sonnei
challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis.
Dissemination ofMycobacterium tuberculosisIs Influenced by Host Factors and Precedes the Initiation of T-Cell Immunity
Authors: Chackerian Alissa A. , Alt Jennifer M. , Perera Thushara V. , Dascher Christopher C. , Behar Samuel M. 
Published: July 2002

ABSTRACTWe report that dissemination ofMycobacterium tuberculosisin the mouse is under host control and precedes the initiation of T-cell immunity. Nine to eleven days after aerosol inoculation,M. tuberculosisdisseminates to the pulmonary lymph nodes (LN), whereM. tuberculosis-specific T cells are detected 2 to 3 days thereafter. This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN. Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance ofM. tuberculosis-specific T cells in the lung and the spleen. In all cases studied, dissemination to the LN or the spleen preceded activation ofM. tuberculosis-specific T cells in that organ. Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response toM. tuberculosissooner than C3H mice generate an immune response. Thus, instead of spreading infection, early dissemination ofM. tuberculosismay aid in the initiation of an appropriate and timely immune response. We hypothesize that this early initiation of immunity following inoculation withM. tuberculosismay contribute to the superior resistance of C57BL/6 mice.
Theomp-1Major Outer Membrane Multigene Family ofEhrlichia chaffeensisIs Differentially Expressed in Canine and Tick Hosts
Authors: Unver Ahmet , Rikihisa Yasuko , Stich Roger W. , Ohashi Norio , Felek Suleyman 
Published: July 2002

ABSTRACTSixteen of 22omp-1paralogs encoding 28-kDa-range immunodominant outer membrane proteins ofEhrlichia chaffeensiswere transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed byE. chaffeensisin three developmental stages ofAmblyomma americanumticks before or afterE. chaffeensistransmission to naïve dogs.
Role of Polyphosphate Kinase in Biofilm Formation by
Porphyromonas gingivalis
Authors: Chen Wen , Palmer Robert J. , Kuramitsu Howard K. 
Published: July 2002

ABSTRACT

In order to assess the role of polyphosphate kinase (PPK) in the physiology of
Porphyromonas gingivalis
, a
ppk
gene mutant, CW120, was constructed and characterized.
P. gingivalis
was demonstrated to synthesize short-chain polyphosphate (polyP) but not long-chain polyP. CW120 failed to survive in the stationary phase as well as the parental cell did, and it was attenuated in biofilm formation on polyvinylchloride and glass surfaces. Furthermore, the complementation by insertion of an intact copy of the
ppk
gene into the mutant CW120 restored its biofilm formation and stationary-phase survival. These results suggest that PPK may be important for incorporation of these organisms into subgingival plaque in the human oral cavity.
Alternative Splicing of
LYT1
Transcripts in
Trypanosoma cruzi
Authors: Manning-Cela Rebeca , González Antonio , Swindle John 
Published: July 2002

ABSTRACT

As a result of alternative
trans
splicing, three distinct
LYT1
mRNAs are produced in
Trypanosoma cruzi
, two encoding the full-length LYT1 protein and the third encoding a truncated LYT1 protein lacking a possible signal sequence. Analysis of the three mRNAs in different developmental forms of the parasite revealed that the alternative processing events were regulated differently during the parasite life cycle.
Functional Analysis and Regulation of the Divergent
spuABCDEFGH-spuI
Operons for Polyamine Uptake and Utilization in
Pseudomonas aeruginosa
PAO1
Authors: Lu Chung-Dar , Itoh Yoshifumi , Nakada Yuji , Jiang Ying 
Published: July 2002

ABSTRACT

A multiple-gene locus for polyamine uptake and utilization was discovered in
Pseudomonas aeruginosa
PAO1. This locus contained nine genes designated
spuABCDEFGHI
(
spu
for spermidine and putrescine utilization). The physiological functions of the
spu
genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn
5
transposon-mediated
spu
knockout mutants. Growth and uptake experiments support that the
spuDEFGH
genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that
spuC
encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although
spuA
and
spuB
mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of
lacZ
fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the
spuABCDEFGH
operon and the divergently transcribed
spuI
gene of unknown function. Since the observed induction effect of agmatine was abolished in an
aguA
mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the
spuA-spuI
divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible
spuA
expression and putrescine aminotransferase (
spuC
) formation in the
cbrB
mutant indicated that the
spu
operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.
Novel DNA-Binding Proteins in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Koksharova Olga A. , Wolk C. Peter 
Published: July 2002

ABSTRACT

As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in
Anabaena
sp. strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from
hepC
, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. Such proteins were purified in four steps from extracts of vegetative cells of
Anabaena
sp. Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the
Anabaena
sp. chromosome. The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome. Insertional mutagenesis of
abp2
and
abp3
blocked expression of
hepC
and
hepA
and prevented heterocyst maturation and aerobic fixation of N
2
.
In Vivo and In Vitro Studies of
Bacillus subtilis
Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway
Authors: Olsson Ulf , Billberg Annika , Sjövall Sara , Al-Karadaghi Salam , Hansson Mats 
Published: July 2002

ABSTRACT

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium
Bacillus subtilis
, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of
B. subtilis
and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in
V
max
. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.
The
fixA
and
fixB
Genes Are Necessary for Anaerobic Carnitine Reduction in
Escherichia coli
Authors: Walt Angelique , Kahn Michael L. 
Published: July 2002

ABSTRACT

In
Escherichia coli
, the use of carnitine as a terminal electron acceptor depends on a functional
caiTABCDE
operon. It had been suggested that the adjacent but divergent
fixABCX
operon is also required for carnitine metabolism, perhaps to provide electrons for carnitine reduction. We have constructed
E. coli fixA
and
fixB
mutants and find that they are unable to reduce carnitine to γ-butyrobetaine under anaerobic conditions.
Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen
Authors: Ge Yong , Vaillancourt Frédéric H. , Agar Nathalie Y. R. , Eltis Lindsay D. 
Published: July 2002

ABSTRACT

Toluate dioxygenase (TADO) of
Pseudomonas putida
mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with
m
-toluate, and each component had a full complement of their respective Fe
2
S
2
centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for
m
-toluate, displaying apparent parameters of
KmA = 9 ± 1 μM
,
k
cat
= 3.9 ± 0.2 s
−1
, and
K

m

O
2
= 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where
K

m

O
2
represents the
K

m

for O
2
and
KmA
represents the
K

m

for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity:
m
-toluate > benzoate ≃ 3-chlorobenzoate >
p
-toluate ≃ 4-chlorobenzoate ≫
o
-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O
2
utilization and yielded the corresponding 1,2-
cis
-dihydrodiol. In contrast, the transformation of
ortho
-substituted benzoates was poorly coupled to O
2
utilization, with >10 times more O
2
being consumed than benzoate. However, the apparent
K

m

of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.
DNA-Binding Activities of the HilC and HilD Virulence Regulatory Proteins ofSalmonella entericaSerovar Typhimurium
Authors: Olekhnovich Igor N. , Kadner Robert J. 
Published: July 2002

ABSTRACTThe HilC and HilD proteins ofSalmonella entericaserovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded onSalmonellapathogenicity island 1 (SPI1) and control expression of thehilAgene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in thehilAandhilCpromoters and to a single region in thehilDpromoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions −92 to −52 of thehilCpromoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. ThehilAandhilCpromoters contain multiple binding sites of each type, whereas thehilDpromoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from thehilAorhilDpromoters using purified proteins in vitro but changed the choice of promoter athilC. These results are consistent with a model derived from analysis oflacZfusions stating that HilC and HilD enhancehilAexpression by counteracting a repressing activity.
Construction, Characterization, and Use of TwoListeria monocytogenesSite-Specific Phage Integration Vectors
Authors: Lauer Peter , Chow Man Yin Nora , Loessner Martin J. , Portnoy Daniel A. , Calendar Richard 
Published: July 2002

ABSTRACTTwo site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction inListeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within thecomKgene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated fromEscherichia coliintoL. monocytogenes, forms stable, single-copy integrants at a frequency of ∼10−4per donor cell, and can be used in theL. monocytogenes1/2 and 4b serogroups. Methods for curing endogenous prophages from thecomKattachment site in 10403S-derived strains were developed. pPL1 was used to introduce thehlyandactAgenes atcomK-attBB′in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on theL. monocytogeneschromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3′ end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNAArggene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.
Functional Characterization of Gne (UDP-
N
-Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of
Yersinia enterocolitica
Serotype O:8
Authors: Bengoechea José Antonio , Pinta Elise , Salminen Tiina , Oertelt Clemens , Holst Otto , Radziejewska-Lebrecht Joanna , Piotrowska-Seget Zofia , Venho Reija , Skurnik Mikael 
Published: July 2002

ABSTRACT

The lipopolysaccharide (LPS) O-antigen of
Yersinia enterocolitica
serotype O:8 is formed by branched pentasaccharide repeat units that contain
N
-acetylgalactosamine (GalNAc),
l
-fucose (Fuc),
d
-galactose (Gal),
d
-mannose (Man), and 6-deoxy-
d
-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called
galE
encodes a UDP-
N
-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed
gne
. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an
Escherichia coli
K-12
galE
mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of
E. coli
GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the
N
-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-
N
-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the
E. coli galE
mutant. Finally, we report that
Yersinia
Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.
Characterization of a
Bacillus subtilis
Thermosensitive Teichoic Acid-Deficient Mutant: Gene
mnaA
(
yvyH
) Encodes the UDP-
N
-Acetylglucosamine 2-Epimerase
Authors: Soldo Blazenka , Lazarevic Vladimir , Pooley Harold M. , Karamata Dimitri 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
thermosensitive mutant ts-21 bears two C-G→T-A transitions in the
mnaA
gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-
N
-acetylglucosamine into UDP-
N
-acetylmannosamine, a precursor of the teichoic acid linkage unit.