bluuebunny/crossref_metadata_2025_split huggingface dataset:
A 60-Kilodalton Immunodominant Glycoprotein Is Essential for Cell Wall Integrity and the Maintenance of Cell Shape in
Streptococcus mutans
Authors: Chia Jean-San , Chang Lan Yi , Shun Chia-Tung , Chang Ying-Ying , Chen Jen-Yang 
Published: July 2002

ABSTRACT

We have demonstrated previously by Western blotting that in naturally sensitized humans, the serum or salivary antibody response to
Streptococcus mutans
was directed predominantly to a protein antigen with a size of approximately 60-kDa. To identify this immunodominant antigen, specific serum antibodies were eluted from immunoblots and five positive clones with inserts ranging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of
Streptococcus mutans
GS-5. Amino acid sequencing established the identity of this immunodominant antigen, a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associated general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide sequence. Discrepancy in the molecular mass was also observed in recombinant his-tagged IDG-60 (rIDG-60) expressed from
Escherichia coli
. Glycosylation, consisting of sialic acid, mannose galactose, and
N
-acetylgalactosamine, was detected by lectin binding to IDG-60 in cell wall extracts from
S. mutans
and rIDG-60 expressed in vivo or translated in vitro. Despite the presence of multiple Asn or Ser or Thr glycosylation sites, IDG-60 was resistant to the effect of
N
-glycosidase F and multiple
O
-glycosidase molecules but not to β-galactosidase. Insertional inactivation of the gene encoding IDG-60,
sagA
, resulted in a retarded growth rate, destabilization of the cell wall, and pleiomorphic cell shape with multifold ingrowth of cell wall. In addition, distinct from the parental GS-5 strain, the isogenic mutant GS-51 was unable to survive the challenge of low pH and high osmotic pressure or high temperature. Expression of the wild-type gene in trans within GS-51 from plasmid pDL277 complemented the growth defect and restored normal cell shape. These results suggested that IDG-60 is essential for maintaining the integrity of the cell wall and the uniformity of cell shape, both of which are indispensable for bacteria survival under stress conditions.
Molecular Characterization of Thermoinduced Immunogenic Proteins Q1p42 and Hsp15 of
Leptospira interrogans
Authors: Nally Jarlath E. , Artiushin Sergey , Timoney John F. 
Published: July 2002

ABSTRACT

Leptospira interrogans
is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts. In vitro studies of organisms cultured at 30°C and shifted to 37°C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J. E. Nally, J. F. Timoney, and B. Stevenson, Infect. Immun. 69:400–404, 2001). In order to specifically identify antigenic proteins upregulated at 37°C, groups of three ponies were immunized with organisms shifted to 37°C for 5 to 7 days or with organisms maintained at 30°C. A lambda ZAP II genomic DNA library was screened with the pool of antisera to organisms shifted to 37°C. Clones reactive with this pool but unreactive with the pool of pony antisera to organisms cultured at 30°C were selected for further analysis. Sequence analysis of the first two clones identified open reading frames for proteins designated Qlp42 and Hsp15. Qlp42 is predicted to be an outer membrane lipoprotein. Its synthesis was upregulated when cultures were shifted from 30 to 37°C and downregulated when cultures were shifted from 37 to 30°C. Although the predicted molecular mass of Qlp42 is 39.8 kDa for the mature protein, Qlp42-specific equine antiserum was reactive with two bands of 30 and 29.5 kDa. Hsp15 is a stress response protein and a member of the Hsp20/α-crystallin family. PCR detected homologues of
qlp
42 and
hsp
15 in pathogenic serovars of
L. interrogans
but not in the nonpathogenic
Leptospira biflexa
. Enzyme-linked immunosorbent assays of antibody in convalescent sera from mares naturally infected with
L. interrogans
suggest that Qlp42 is expressed during leptospiral infection.
Anti-Class II Monoclonal Antibody-TargetedVibrio choleraeTcpA Pilin: Modulation of Serologic Response, Epitope Specificity, and Isotype
Authors: Wu Jia-Yan , Taylor Ronald K. , Wade William F. 
Published: July 2002

ABSTRACTToxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.
Coiling Phagocytosis ofBorrelia burgdorferiby Primary Human Macrophages Is Controlled by CDC42Hs and Rac1 and Involves Recruitment of Wiskott-Aldrich Syndrome Protein and Arp2/3 Complex
Authors: Linder Stefan , Heimerl Christiane , Fingerle Volker , Aepfelbacher Martin , Wilske Bettina 
Published: July 2002

ABSTRACTLyme borreliosis is a multisystemic disorder primarily affecting the skin, nervous system, and joints. It is caused by the spirocheteBorrelia burgdorferisensu lato and is transmitted via ticks of theIxodidaefamily. Persistence of borreliae within macrophages has been implicated in the often chronic history of borreliosis. The uptake ofB. burgdorferiby professional phagocytes occurs predominantly by coiling phagocytosis, a host cell-driven process in which single pseudopods wrap around and engulf the spirochetes. In the present study, we investigated the molecular machinery and the signal transduction pathways controlling the formation of these unique uptake structures. We found that the phagocytosis of borreliae by primary human macrophages is accompanied by the formation of f-actin-rich structures, which in their morphological organization correspond well to the earlier described coiling pseudopods. Further experiments revealed that Wiskott-Aldrich Syndrome protein and Arp2/3 complex, major regulators of actin polymerization, are also recruited to these sites of actin accumulation. In addition, inhibition of an upstream regulator of Wiskott-Aldrich Syndrome protein, the Rho-family GTPase CDC42Hs, greatly inhibited the occurrence of borrelia-induced phagocytic uptake structures. Inhibition of Rac1, another Rho family GTPase, had a less-pronounced inhibitory effect, while blocking of Rho activity showed no discernible influence. These results suggest that basic mechanisms of actin polymerization that control other types of phagocytosis are also functional in the formation of the morphologically unique uptake structures in coiling phagocytosis. Our findings should enhance the understanding of the infection process ofB. burgdorferiand contribute to devising new strategies for countering Lyme disease.
Induction and Distribution of Intestinal Immune Responses after Administration of Recombinant Cholera Toxin B Subunit in the Ileal Pouches of Colectomized Patients
Authors: Kilhamn Jan , Brevinge Hans , Quiding-Järbrink Marianne , Svennerholm Ann-Mari , Jertborn Marianne 
Published: July 2002

ABSTRACT
The induction and dissemination of mucosal immune responses to recombinant cholera toxin B subunit (rCTB) administered into the ileal pouches of patients, who had been colectomized because of ulcerative colitis, was analyzed. Biopsies from the duodenum and ileal pouch were collected, along with peripheral blood and ileostomy fluids. Two immunizations induced strong CTB-specific immunoglobulin A (IgA) antibody-secreting cell (ASC) responses in the duodenum in five of five patients, whereas weaker and less-frequent ASC responses were noted in the ileal pouch. Intestine-derived CTB-specific IgA ASCs were found in peripheral blood in three of the five patients. The vaccination also induced significant IgA antitoxin titer rises in ileostomy fluid in all of the patients. Increased production of gamma interferon in cell cultures from the ileal pouch was found in four of five patients after the vaccination. These results clearly indicate that rCTB administered into the distal ileum is capable of inducing B-cell responses in the “entire” small intestine and that homing of immunocompetent cells occurs preferentially to the duodenum.
Purification and Characterization of
Borrelia burgdorferi
from Feeding Nymphal Ticks (
Ixodes scapularis
)
Authors: Rathinavelu Sivaprakash , de Silva Aravinda M. 
Published: July 2002

ABSTRACT

Here we describe a protocol for purifying
Borrelia burgdorferi
from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels of
B. burgdorferi
outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.
CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by RecombinantListeria monocytogenesduring Secondary Infection
Authors: Tvinnereim Amy R. , Hamilton Sara E. , Harty John T. 
Published: July 2002

ABSTRACTUnderstanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuatedactA-deficientListeria monocytogenes, on the CD8+-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinantL. monocytogenes. Challenges of immune mice withactA-deficient and with wild-type recombinantL. monocytogenesgenerated similar numbers of CD8+T cells specific for the NP118-126 epitope. High-dose immunization withactA-deficientL. monocytogenesresulted in substantial numbers of CD8+T cells specific for theL. monocytogenesLLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinantL. monocytogenesresulted in rapid control of the infection and decreased CD8+-T-cell responses against both the secreted and nonsecreted form of the recombinant antigen compared to the response of naïve mice. In contrast, mice immunized with a low dose ofactA-deficientL. monocytogeneshad ∼10-fold fewer effector and memory T cells specific for LLO91-99 and a substantially higher CD8+-T-cell response against the recombinant antigen after challenge with recombinantL. monocytogenes. Although mice immunized with low-doseactA-deficientL. monocytogeneshad a substantial recall response to LLO91-99, which reached the same levels by 5 to 7 days postchallenge as that in high-dose-immunized mice, they exhibited decreased ability to controlL. monocytogenesreplication. Thus, the level of antivector immunity impacts the control of infection and efficiency of priming responses against new antigens introduced with the same vector.
A Complex Insertion Sequence Cluster at a Point of Interaction between the Linear Plasmid SCP1 and the Linear Chromosome of
Streptomyces coelicolor
A3(2)
Authors: Yamasaki Masayuki , Miyashita Kiyotaka , Cullum John , Kinashi Haruyasu 
Published: July 2002

ABSTRACT

The giant linear plasmid SCP1 can integrate into the central region of the linear chromosome of
Streptomyces coelicolor
A3(2). Nucleotide sequence analysis around the target site for SCP1 integration in strain M145 identified a total of five copies of four insertion sequences (ISs) in a 6.5-kb DNA stretch. Three of the four (IS
468
, IS
469
, and IS
470
) are new IS elements, and the other is IS
466
. All of these elements contain one open reading frame which encodes a transposase-like protein. Two copies of IS
468
(IS
468
A and -B) are tandemly aligned at the left end of the cluster. Following these, IS
469
and IS
466
are located in a tail-to-tail orientation with 69.3% identity to each other. IS
470
is located at the right end of the cluster. The activities of IS
466
and IS
468
were demonstrated by transposition experiments and sequence comparison of several copies, respectively.
The Length of the Combined 3′ Untranslated Region and Poly(A) Tail Does Not Control Rates of Glyceraldehyde-3-Phosphate Dehydrogenase mRNA Translation in Three Species of Parasitic Protists
Authors: ter Kuile Benno H. , Sallés Fernando J. 
Published: July 2002

ABSTRACT

Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3′ end has a role in regulating rates of translation in the parasitic protists
Trypanosoma brucei
,
Leishmania donovani
, and
Trichomonas vaginalis
. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3′ end under different growth conditions in all three species. Our results showed that the combined 3′ untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.
Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2
Authors: Rosche Thomas M. , Siddique Azeem , Larsen Michelle H. , Figurski David H. 
Published: July 2002

ABSTRACT

Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci:
trfA
, the replication initiator gene, and
oriV
, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the
incC
gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of
incC
(IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for
incC
-mediated incompatibility is a KorB-binding site (O
B
). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the
incC
and
korB
genes individually has no obvious effect on
Escherichia coli
cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing
incC
,
korB
, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an
incC
-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.
Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium
Pseudomonas
Strain B10: Identification and Functional Analysis of the
l
-Ornithine

N
5

-Oxygenase (
psbA
) Gene
Authors: Ambrosi C. , Leoni L. , Putignani L. , Orsi N. , Visca P. 
Published: July 2002

ABSTRACT

Pseudobactin
B10
, the fluorescent siderophore produced by the rhizobacterium
Pseudomonas
strain B10, contains the hydroxamate ligand
d
-

N
5

-hydroxyornithine (
d
-

N
5

-OH-Orn). We cloned the
l
-Orn

N
5

-oxygenase (
psbA
) gene from a genomic library of
Pseudomonas
strain B10 and demonstrated that PsbA is involved in the conversion of
l
-Orn to its

N
5

-OH derivative. PsbA shows significant similarity to microbial ω-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The
psbA
gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific
psbA
mutant of
Pseudomonas
strain B10 was biochemically complemented with the precursor
l
-

N
5

-OH-Orn, suggesting that
l
-Orn is hydroxylated before conversion to the
d
isomer. The
l
-Orn

N
5

-hydroxylase-defective mutants of
Pseudomonas
strain B10 and
Pseudomonas aeruginosa
PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen
Erwinia carotovora
in iron-poor medium. The extent of in vitro inhibition of
E. carotovora
was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.
Exfoliatin-Producing Strains Define a Fourth
agr
Specificity Group in
Staphylococcus aureus
Authors: Jarraud S. , Lyon G. J. , Figueiredo A. M. S. , Gérard Lina , Vandenesch F. , Etienne J. , Muir T. W. , Novick R. P. 
Published: July 2002

ABSTRACT

The staphylococcal virulon is activated by the density-sensing
agr
system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by
agrD
. A central segment of the
agr
locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of
Staphylococcus aureus
isolates into three different
agr
specificity groups and to the division of non-
aureus
staphylococci into a number of others. The AIPs cross-inhibit the
agr
responses between groups. We have previously shown that most menstrual toxic shock strains belong to
agr
specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth
agr
specificity group among
S. aureus
strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the
agr
response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the
agr
autoinduction threshold.
Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς
S
-Dependent Transcription in
Pseudomonas putida
Authors: Ojangu Eve-Ly , Tover Andres , Teras Riho , Kivisaar Maia 
Published: July 2002

ABSTRACT

The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ς
S
. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ς
S
(Eς
S
) recognizes a number of promoters which are also recognized by that containing ς
70
(Eς
70
). We have previously shown that transposon Tn
4652
can activate silent genes in starving
Pseudomonas putida
cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς
70
-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and
rpoS
-deficient mutant
P. putida
, the effect of ς
S
on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ς
S
-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ς
S
-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ς
S
-specific transcription in concert with the sequence downstream from the −10 box.
On the Functional Interchangeability, Oxidant versus Reductant, of Members of the Thioredoxin Superfamily
Authors: Debarbieux Laurent , Beckwith Jon 
Published: July 2002

ABSTRACT

Escherichia coli
thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Åslund, and J. Beckwith, EMBO J. 17:5543–5550, 1998). We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L. Debarbieux and J. Beckwith, Proc. Natl. Acad. Sci. USA 95:10751–10756, 1998). Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it. While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself. Further, we confirm the findings of Jonda et al. (S. Jonda, M. Huber-Wunderlich, R. Glockshuber, and E. Mössner, EMBO J. 18:3271–3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex. Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant.
Stress Triggers a Process That Limits Activation of the
Bacillus subtilis
Stress Transcription Factor ς
B
Authors: Scott Janelle M. , Mitchell Theresa , Haldenwang W. G. 
Published: July 2002

ABSTRACT

Stress-induced activation of the
Bacillus subtilis
transcription factor ς
B
is transitory. To determine whether the process that limits ς
B
activation is itself triggered by stress,
B. subtilis
strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of ς
B
activity. Without ethanol treatment, the induced cultures displayed continuously high ς
B
activity. Ethanol treatment restricted ongoing ς
B
activity, but only in strains with intact
rsbX
and -
S
genes. The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect. The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict ς
B
induction following stress.