bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antigenic Specificity of theMycobacterium lepraeHomologue of ESAT-6
Authors: Spencer John S. , Marques Maria Angela M. , Lima Monica C. B. S. , Junqueira-Kipnis Ana Paula , Gregory Bruce C. , Truman Richard W. , Brennan Patrick J. 
Published: July 2002

ABSTRACTThe sequence of theMycobacterium lepraehomologue of ESAT-6 shows only 36% amino acid correspondence to that fromMycobacterium tuberculosis. Anti-M. lepraeESAT-6 polyclonal and monoclonal antibodies and T-cell hybridomas reacted only with the homologous protein and allowed identification of the B- and T-cell epitopes. The protein is expressed inM. lepraeand appears in the cell wall fraction. Thus,M. lepraeESAT-6 shows promise as a specific diagnostic agent for leprosy.
Role of Glutathione Metabolism of
Treponema denticola
in Bacterial Growth and Virulence Expression
Authors: Chu Lianrui , Dong Zheng , Xu Xiaoping , Cochran David L. , Ebersole Jefferey L. 
Published: July 2002

ABSTRACT

Hydrogen sulfide (H
2
S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis.
Treponema denticola
, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H
2
S. The metabolic pathway leading to H
2
S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for
T. denticola
to produce H
2
S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H
2
S production by this microorganism. In addition to H
2
S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H
2
S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in
T. denticola
responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56°C) and freezing (−20°C), by sonication, and by exposure to
N
α-
p
-tosyl-
l
-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete
T. denticola
; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.
Peripheral Blood Mononuclear Cell Activation Induced byLeptospira interrogansGlycolipoprotein
Authors: Diament Decio , Brunialti Milena Karina Colo , Romero Eliete Calo , Kallas Esper Georges , Salomao Reinaldo 
Published: July 2002

ABSTRACTLeptospira interrogansglycolipoprotein (GLP) has been implicated in pathological and functional derangement seen in leptospirosis. The goal of this study was to evaluate GLP's ability to induce cellular activation, as assessed by cytokine production and expression of surface activation markers. GLP extracted from either pathogenicL. interrogansserovar Copenhageni or nonpathogenicLeptospira biflexaserovar Patoc (GLPp) was used to stimulate peripheral blood mononuclear cell cultures from healthy donors. Supernatant cytokine levels were measured by enzyme-linked immunosorbent assay. Expression of CD69 and HLA-DR on lymphocytes and monocytes, as well as lipopolysaccharide (LPS) binding, were measured by flow cytometry. At 6 h of incubation, GLP induced a significant rise in tumor necrosis factor alpha levels, which dropped progressively until 72 h of incubation. Interleukin-10 peak levels were obtained at between 24 and 48 h, with sustained levels until 72 h of incubation. The response magnitude was proportional to the GLP dose. CD69 expression on T lymphocytes and monocytes increased significantly, as did HLA-DR expression on monocytes. GLPp induced no CD69 or HLA-DR expression. GLP did not block biotinylated LPS binding to monocytes, suggesting that different pathways are used to induce cell activation. In conclusion, GLP induces cellular activation and may play a major role in the pathogenesis of leptospirosis.
Coexpression of Interleukin-12 Chains by a Self-Splicing Vector Increases the Protective Cellular Immune Response of DNA andMycobacterium bovisBCG Vaccines againstMycobacterium tuberculosis
Authors: Palendira Umaimainthan , Kamath Arun T. , Feng Carl G. , Martin Ela , Chaplin Paul J. , Triccas James A. , Britton Warwick J. 
Published: July 2002

ABSTRACTMore effective vaccines againstMycobacterium tuberculosismay contribute to the control of this major human pathogen. DNA vaccines encoding single mycobacterial proteins stimulate antimycobacterial T-cell responses and induce partial protection againstM. tuberculosisin animal models. The protective efficacy of these vaccines encoding a single antigen, however, has been less than that afforded by the current vaccine,Mycobacterium bovisbacillus Calmette-Guérin (BCG). The heterodimeric cytokine interleukin-12 (IL-12) potentiates the induction and maintenance of the type 1 helper T-cell response. We have developed a novel self-splicing vector based on the 2A protein of foot-and-mouth disease virus that permits the coordinate expression of both chains of IL-12 (p2AIL12). Coimmunization with this vector and DNA expressingM. tuberculosisantigen 85B or MPT64 enhanced the specific lymphocyte proliferative response and increased the frequency of specific gamma interferon-secreting T cells against the whole protein and a defined CD8+T-cell epitope on MPT64. Further, coimmunizing with p2AIL12 significantly increased the protective efficacy of DNA-85 in the lung against an aerosol challenge withM. tuberculosisto the level achieved with BCG. Therefore, codelivery of an IL-12-secreting plasmid may be a potent strategy for enhancing the protective efficacy of vaccines againstM. tuberculosis.
RecombinantOchrobactrum anthropiExpressingBrucella abortusCu,Zn Superoxide Dismutase Protects Mice againstB. abortusInfection Only after Switching of Immune Responses to Th1 Type
Authors: He Yongqun , Vemulapalli Ramesh , Schurig Gerhardt G. 
Published: July 2002

ABSTRACTThe members of the genusBrucellaare gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans. Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use. Here we report that a bacterium that is closely related toBrucellaspecies,Ochrobactrum anthropi, can be used as a vaccine vector for the delivery ofBrucellaantigens to mice, leading to the elicitation of protective immunity against brucellosis.Brucella abortusCu,Zn superoxide dismutase (SOD), a protectiveBrucellaantigen, was expressed in large amounts inO. anthropistrain 49237 by use of the broad-host-range plasmid pBBR1MCS. NeitherO. anthropistrain 49237 nor the recombinantO. anthropistrain 49237SOD, expressingB. abortusCu,Zn SOD, provided protection against virulentBrucellainfection in mice. Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice. After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, bothO. anthropistrain 49237 and the recombinantO. anthropistrain 49237SOD induced protection in mice. However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237.
Modulation of
Borrelia burgdorferi
Stringent Response and Gene Expression during Extracellular Growth with Tick Cells
Authors: Bugrysheva Julia , Dobrikova Elena Y. , Godfrey Henry P. , Sartakova Marina L. , Cabello Felipe C. 
Published: July 2002

ABSTRACT

Borrelia burgdorferi
N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support
B. burgdorferi
N40 growth. Growth of
B. burgdorferi
N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in
relA
/
spoT
mRNA, an eightfold net decrease in
bmpD
mRNA, and a fourfold increase in
rpsL-bmpD
mRNA compared to growth of
B. burgdorferi
in BSK-H medium. As a result, the polycistronic
rpsL-bmpD
mRNA level increased from 3 to 100% of the total
bmpD
message. These observations demonstrate that there are reciprocal interactions between
B. burgdorferi
and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in
B. burgdorferi
growing in BSK-H medium is ameliorated in
B. burgdorferi
growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of
B. burgdorferi
with tick cells in vitro when it is coupled with genetic methods to isolate and complement
B. burgdorferi
mutants.
Chlamydia trachomatis
Induces Remodeling of the Actin Cytoskeleton during Attachment and Entry into HeLa Cells
Authors: Carabeo Reynaldo A. , Grieshaber Scott S. , Fischer Elizabeth , Hackstadt Ted 
Published: July 2002

ABSTRACT

To elucidate the host cell machinery utilized by
Chlamydia trachomatis
to invade epithelial cells, we examined the role of the actin cytoskeleton in the internalization of chlamydial elementary bodies (EBs). Treatment of HeLa cells with cytochalasin D markedly inhibited the internalization of
C. trachomatis
serovar L2 and D EBs. Association of EBs with HeLa cells induced localized actin polymerization at the site of attachment, as visualized by either phalloidin staining of fixed cells or the active recruitment of GFP-actin in viable infected cells. The recruitment of actin to the specific site of attachment was accompanied by dramatic changes in the morphology of cell surface microvilli. Ultrastructural studies revealed a transient microvillar hypertrophy that was dependent upon
C. trachomatis
attachment, mediated by structural components on the EBs, and cytochalasin D sensitive. In addition, a mutant CHO cell line that does not support entry of
C. trachomatis
serovar L2 did not display such microvillar hypertrophy following exposure to L2 EBs, which is in contrast to infection with serovar D, to which it is susceptible. We propose that
C. trachomatis
entry is facilitated by an active actin remodeling process that is induced by the attachment of this pathogen, resulting in distinct microvillar reorganization throughout the cell surface and the formation of a pedestal-like structure at the immediate site of attachment and entry.
Isolation and Characterization of Nonchemotactic CheZ Mutants of
Escherichia coli
Authors: Boesch Kristin C. , Silversmith Ruth E. , Bourret Robert B. 
Published: July 2002

ABSTRACT

The
Escherichia coli
CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected
cheZ
to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of
cheZ
, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ
1–189
peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.
The Flagellar Hook Protein, FlgE, of
Salmonella enterica
Serovar Typhimurium Is Posttranscriptionally Regulated in Response to the Stage of Flagellar Assembly
Authors: Bonifield Heather R. , Yamaguchi Shigeru , Hughes Kelly T. 
Published: July 2002

ABSTRACT

We investigated the posttranscriptional regulation of
flgE
, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the
flgE
gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook–basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly.
Recombination Is Essential for Viability of an
Escherichia coli dam
(DNA Adenine Methyltransferase) Mutant
Authors: Marinus M. G. 
Published: July 2002

ABSTRACT

Double mutants of
Escherichia coli dam
(DNA adenine methyltransferase) strains with
ruvA
,
ruvB
, or
ruvC
could not be constructed, whereas
dam
derivatives with
recD
,
recF
,
recJ
, and
recR
were viable. The
ruv
gene products are required for Holliday junction translocation and resolution of recombination intermediates. A
dam recG
(Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a
dam lexA
(Ind
−
) host was abrogated by the simultaneous presence of plasmids encoding both
recA
and
ruvAB
. This result indicates that of more than 20 SOS genes, only
recA
and
ruvAB
need to be derepressed to allow for
dam
mutant survival. The presence of
mutS
or
mutL
mutations allowed the construction of
dam lexA
(Ind
−
) derivatives. The requirement for
recA
,
recB
,
recC
,
ruvA
,
ruvB
,
ruvC
, and possibly
recG
gene expression indicates that recombination is essential for viability of
dam
bacteria probably to repair DNA double-strand breaks. The effect of
mutS
and
mutL
mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of
dam
cells to certain DNA-damaging agents.
Cell Cycle Arrest in Archaea by the Hypusination Inhibitor
N
1
-Guanyl-1,7-Diaminoheptane
Authors: Jansson B. P. Mattias , Malandrin Laurence , Johansson Hans E. 
Published: July 2002

ABSTRACT

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor
N
1
-guanyl-1,7-diaminoheptane (GC
7
) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC
7
, whereas the bacterium
Escherichia coli
is not, (ii) GC
7
causes rapid and reversible arrest of growth of the archaeon
Sulfolobus acidocaldarius
, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.
Streptococcus gordonii
Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes
Authors: Loo C. Y. , Corliss D. A. , Ganeshkumar N. 
Published: July 2002

ABSTRACT

Viridans streptococci, which include
Streptococcus gordonii
, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of
S. gordonii
Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn
916
transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of
S. gordonii
Challis were identified based on Southern hybridization with a Tn
916
-based probe and DNA sequences of the Tn
916
-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.
Characterization of a 12-Kilodalton Rhodanese Encoded by
glpE
of
Escherichia coli
and Its Interaction with Thioredoxin
Authors: Ray W. Keith , Zeng Gang , Potters M. Benjamin , Mansuri Aqil M. , Larson Timothy J. 
Published: July 2002

ABSTRACT

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from
Escherichia coli
. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The
glpE
gene, a member of the
sn
-glycerol 3-phosphate (
glp
) regulon of
E. coli
, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The

K
m

s for SSO
3
2−
and CN
−
were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a
k
cat
of 230 s
−1
. Thioredoxin 1 from
E. coli
, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent

K
m

of 34 μM when thiosulfate was near its

K
m

, suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (∼17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (
http://www.ncbi.nlm.nih.gov/COG/
) for which sulfurtransferase activity has been confirmed.
Identification of SopE2, a
Salmonella
Secreted Protein Which Is Highly Homologous to SopE and Involved in Bacterial Invasion of Epithelial Cells
Authors: Bakshi C. S. , Singh V. P. , Wood M. W. , Jones P. W. , Wallis T. S. , Galyov E. E. 
Published: July 2002

ABSTRACT

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying
Salmonella
pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the
sopE2
gene which is present and conserved in pathogenic strains of
Salmonella
. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.
Divergence of Eukaryotic Secretory Components: the
Candida albicans
Homolog of the
Saccharomyces cerevisiae
Sec20 Protein Is N Terminally Truncated, and Its Levels Determine Antifungal Drug Resistance and Growth
Authors: Weber Yvonne , Santore Uwe J. , Ernst Joachim F. , Swoboda Rolf K. 
Published: July 2002

ABSTRACT

Sec20p is a component of the yeast
Saccharomyces cerevisiae
secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen
Candida albicans
. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to
S. cerevisiae
Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both
C. albicans SEC20
alleles could not be constructed, we placed
SEC20
under transcriptional control of two regulatable promoters,
MET3p
and
PCK1p
. Repression of
SEC20
expression in these strains prevented (
MET3p-SEC20
allele) or retarded (
PCK1p-SEC20
allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced
SEC20
expression in the
PCK1p-SEC20
strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than
S. cerevisiae
, but the lack of the N-terminal domain and the apparent absence of a close
TIP20
homolog in the
C. albicans
genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.