bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calprotectin Expression In Vitro by Oral Epithelial Cells Confers Resistance to Infection byPorphyromonas gingivalis
Authors: Nisapakultorn K. , Ross K. F. , Herzberg M. C. 
Published: July 2002

ABSTRACTCalprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression.Porphyromonas gingivaliswas permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalizedP. gingivalisthan sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure toP. gingivalisinduced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. UsingP. gingivalisprotease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion byP. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance toP. gingivalisinfection.
Role of
Enterococcus faecalis
Surface Protein Esp in the Pathogenesis of Ascending Urinary Tract Infection
Authors: Shankar Nathan , Lockatell C. Virginia , Baghdayan Arto S. , Drachenberg C. , Gilmore Michael S. , Johnson David E. 
Published: July 2002

ABSTRACT

Enterococcus faecalis
bacteria isolated from patients with bacteremia, endocarditis, and urinary tract infections more frequently express the surface protein Esp than do fecal isolates. To assess the role of Esp in colonization and persistence of
E. faecalis
in an animal model of ascending urinary tract infection, we compared an Esp
+
strain of
E. faecalis
to its isogenic Esp-deficient mutant. Groups of CBA/J mice were challenged transurethrally with 10
8
CFU of either the parent or mutant strain, and bacteria in the urine, bladder, and kidneys were enumerated 5 days postinfection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the parent strain than from the bladder and urine of mice challenged with the mutant. Colonization of the kidney, however, was not significantly different between the parent and mutant strains. Histopathological evaluations of kidney and bladder tissue done at 5 days postinfection did not show marked histopathological changes consistent with inflammation, mucosal hyperplasia, or apoptosis, and there was no observable difference between the mice challenged with the parent and those challenged with the mutant. We conclude that, while Esp does not influence histopathological changes associated with acute urinary tract infections, it contributes to colonization and persistence of
E. faecalis
at this site.
Plasmidic versus Insertional Cloning of Heterologous Genes inMycobacterium bovisBCG: Impact on In Vivo Antigen Persistence and Immune Responses
Authors: Méderlé I. , Bourguin I. , Ensergueix D. , Badell E. , Moniz-Peireira J. , Gicquel B. , Winter N. 
Published: July 2002

ABSTRACTBivalent recombinant strains ofMycobacterium bovisBCG (rBCG) expressing the early regulatorynefand the structuralgag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of bothnefandgagin the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of thenef-gagoperon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, thenef-gagoperon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.
Influence of the Length of the Lipooligosaccharide α Chain on Its Sialylation in
Neisseria meningitidis
Authors: Tsai Chao-Ming , Kao George , Zhu Peixuan 
Published: July 2002

ABSTRACT

The sialylation of lipooligosaccharide (LOS) in
Neisseria meningitidis
plays a role in the resistance of the organism to killing by normal human serum. The length of the α chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of
N. meningitidis
LOS in vitro and in vivo. The α chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galβ1-4Glc) in the α chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.
Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A
Authors: Masri Heather P. , Cornelissen Cynthia Nau 
Published: July 2002

ABSTRACT

The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in
Escherichia coli
, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.
Induction ofdnaKthrough Its Native Heat Shock Promoter Is Necessary for Intramacrophagic Replication ofBrucella suis
Authors: Köhler Stephan , Ekaza Euloge , Paquet Jean-Yves , Walravens Karl , Teyssier Jacques , Godfroid Jacques , Liautard Jean-Pierre 
Published: July 2002

ABSTRACTThe heat shock protein DnaK is essential for intramacrophagic replication ofBrucella suis. The replacement of the stress-inducible, nativednaKpromoter ofB. suisby the promoter of the constitutively expressedblagene resulted in temperature-independent synthesis of DnaK. In contrast to adnaKnull mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutivednaKmutant, which showed high sensitivity to H2O2-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction ofdnaKis a crucial event in the intracellular replication ofB. suis.
Randomized, Controlled Human Challenge Study of the Safety, Immunogenicity, and Protective Efficacy of a Single Dose of Peru-15, a Live Attenuated Oral Cholera Vaccine
Authors: Cohen Mitchell B. , Giannella Ralph A. , Bean Judy , Taylor David N. , Parker Susan , Hoeper Amy , Wowk Stephen , Hawkins Jennifer , Kochi Sims K. , Schiff Gilbert , Killeen Kevin P. 
Published: July 2002

ABSTRACT

Peru-15 is a live attenuated oral vaccine derived from a
Vibrio cholerae
O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 × 10
8
CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10
5
CFU of virulent
V. cholerae
O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (≥3,000 g of diarrheal stool) (
P
= 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (
P
< 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool
V. cholerae
excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.
Role of Tumor Necrosis Factor Alpha inHelicobacter pyloriGastritis in Tumor Necrosis Factor Receptor 1-Deficient Mice
Authors: Thalmaier Ulrike , Lehn Norbert , Pfeffer Klaus , Stolte Manfred , Vieth Michael , Schneider-Brachert Wulf 
Published: July 2002

ABSTRACTIncreased gastric production of interleukin 8 and tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis ofHelicobacter pylori-associated gastroduodenal disease. In the present study we used a mouse model to demonstrate whether loss of the tumor necrosis factor receptor 1 (TNF-R1) function leads to differences in gastric inflammation or the systemic immune response inH. pyloriinfection. Six different clinical isolates ofH. pylori(three cytotoxin-positive and three cytotoxin-negative strains) were adapted to C57BL/6 mice. TNF-R1-deficient (TNF-R1−/−) mice (n= 19) and isogenetic controls (n= 24) were infected and sacrificed after 4 weeks of infection. Inflammation of the stomach and the humoral immune response toH. pyloriwere evaluated by histological, immunohistochemical, and serological methods. There was no detectable difference in the grade or activity of gastritis in TNF-R1−/−mice when they were compared with wild-type mice, but the number of lymphoid aggregates was slightly reduced in the gastric mucosa of TNF-R1−/−mice. Interestingly, total immunoglobulin G (IgG), as well as IgG1, IgG2b, and IgG3,H. pylori-specific antibody titers were significantly higher in wild-type mice. As revealed by immunoblot analysis, the difference in reactivity againstH. pyloriantigens was not based on a failure to recognize singleH. pyloriantigens in TNF-R1−/−mice. We therefore suggest that TNF-R1-mediated TNF-α signals might support a systemic humoral immune response againstH. pyloriand that the gastric inflammatory response toH. pyloriinfection seems to be independent of TNF-R1-mediated signals.
Modulation of C5aR Expression on Human Neutrophils by Encapsulated and AcapsularCryptococcus neoformans
Authors: Monari Claudia , Kozel Thomas R. , Bistoni Francesco , Vecchiarelli Anna 
Published: July 2002

ABSTRACTCryptococcus neoformansand cryptococcal surface polysaccharides influenced C5aR expression on human polymorphonuclear neutrophils (PMN). Encapsulated and acapsular strains produced dramatically different effects. Treatment of PMN with acapsular cryptococci up-regulated C5aR expression; whereas treatment with encapsulated cells suppressed C5aR expression. Glucuronoxylomannan (GXM), the principal constituent of the cryptococcal capsule, was responsible for such inhibition. Increased C5aR expression following treatment with acapsular cryptococci was accompanied by increased binding of C5a to PMN, increased superoxide production in response to stimulation with C5a, and an increased chemotactic response to C5a. Conversely, decreased C5aR expression following treatment with encapsulated cryptococci or acapsular cryptococci that had been pretreated with GXM was accompanied by decreased binding of C5a to PMN and a decreased chemotactic response to C5a. Our results raise the possibility that the down-regulation of C5aR expression by encapsulated cryptococci might alter PMN function at the site of cryptococcal infection.
Natural Diversity in the N Terminus of the Mature Vacuolating Cytotoxin of
Helicobacter pylori
Determines Cytotoxin Activity
Authors: Letley D. P. , Atherton J. C. 
Published: July 2002

ABSTRACT

Naturally occurring noncytotoxic
vacA
type s2 strains of
Helicobacter pylori
have a 12-residue extension to the vacuolating cytotoxin (VacA) compared with cytotoxic type s1 strains. We show that adding the region encoding this extension to type s1
vacA
completely abolishes vacuolating cytotoxin activity but has no effect on VacA production.
Control of Sporulation Gene Expression in
Bacillus subtilis
by the Chromosome Partitioning Proteins Soj (ParA) and Spo0J (ParB)
Authors: Quisel John D. , Grossman Alan D. 
Published: July 2002

ABSTRACT

Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB), regulate the initiation of sporulation in
Bacillus subtilis
. In a
spo0J
null mutant, sporulation is inhibited by the action of Soj. Soj negatively regulates expression of several sporulation genes by binding to the promoter regions and inhibiting transcription. All of the genes known to be inhibited by Soj are also activated by the phosphorylated form of the transcription factor Spo0A (Spo0A∼P). We found that, in a
spo0J
null mutant, Soj affected sporulation, in part, by decreasing the level of Spo0A protein. Soj negatively regulated transcription of
spo0A
and associated with the
spo0A
promoter region in vivo. Expression of
spo0A
from a heterologous promoter in a
spo0J
null mutant restored Spo0A levels and partly bypassed the sporulation and gene expression defects. Soj did not appear to significantly affect phosphorylation of Spo0A. Thus, in the absence of Spo0J, Soj inhibits sporulation and sporulation gene expression by inhibiting accumulation of the activator protein Spo0A and by acting downstream of Spo0A to inhibit gene expression directly.
Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EII
Glc
) Component of the
Escherichia coli
Phosphotransferase System
Authors: Notley-McRobb Lucinda , Ferenci Thomas 
Published: July 2002

ABSTRACT

Escherichia coli
adapted to glucose-limited chemostats contained mutations in
ptsG
resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII
Glc
) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased
ptsG
transcription in the absence of glucose. Increased expression of wild-type EII
Glc
was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII
Glc
. The conformation of EII
Glc
elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of
pts
genes.
Expression of a Constitutively Active Cdc42 Homologue Promotes Development of Sclerotic Bodies but Represses Hyphal Growth in the Zoopathogenic Fungus
Wangiella
(
Exophiala
)
dermatitidis
Authors: Ye Xiangcang , Szaniszlo Paul J. 
Published: July 2002

ABSTRACT

In contrast to the
CDC42
homologues of
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
, the
WdCDC42
gene in the human pathogenic fungus
Wangiella
(
Exophiala
)
dermatitidis
was found to be nonessential for cell viability. Expression of the constitutively active allele
wdcdc42
G14V
at 37°C induced nonpolarized growth that led to cell enlargement and multiple nucleation. The swollen cells subsequently converted into planate divided bicellular forms or multiply septated sclerotic bodies in post-log phase, when the G14V-altered protein was diminished. The
wdcdc42
G14V
mutation also strongly repressed filamentous growth both in the wild-type strain and in the temperature-sensitive hyphal-form mutant Hf1. In contrast, overexpression of the dominant negative alleles
wdcdc42
T19N
and
wdcdc42
D120A
had no obvious effect on fungal-cell polarization. These results suggested that WdCdc42p plays a unique regulatory role in cellular morphogenesis in
W. dermatitidis
. Activation of this protein in response to extracellular or intracellular signals seems to commit its yeast-like cells to a phenotype transition that produces sclerotic bodies while repressing hyphal development.
Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of
Bacteroides thetaiotaomicron
Authors: Shipman Joseph A. , Berleman James E. , Salyers Abigail A. 
Published: July 2002

ABSTRACT

Bacteroides thetaiotaomicron
, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of
14
C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.
Role of TnrA in Nitrogen Source-Dependent Repression of
Bacillus subtilis
Glutamate Synthase Gene Expression
Authors: Belitsky Boris R. , Wray Lewis V. , Fisher Susan H. , Bohannon Dian E. , Sonenshein Abraham L. 
Published: July 2002

ABSTRACT

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In
Bacillus subtilis
, the
gltAB
operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the
gltAB
operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the
gltAB
promoter. As is true for other genes, the activity of TnrA at the
gltAB
promoter was antagonized by glutamine synthetase under certain growth conditions.