bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role for Fimbriae and Lysine-Specific Cysteine Proteinase Gingipain K in Expression of Interleukin-8 and Monocyte Chemoattractant Protein inPorphyromonas gingivalis-Infected Endothelial Cells
Authors: Nassar Hamdy , Chou Hsin-Hua , Khlgatian Mary , Gibson Frank C. , Dyke T. E. Van , Genco Caroline Attardo 
Published: July 2002

ABSTRACTRecent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogenPorphyromonas gingivalisis capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions ofP. gingivaliswith endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences ofP. gingivalisinfection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition ofP. gingivalisfimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with liveP. gingivalisstrain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherentP. gingivalis fimAmutant DPG3 or whenP. gingivaliswas preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment ofP. gingivalis-infected HUVEC with cytochalsin D, which preventedP. gingivalisinvasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC withE. coliLPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with liveP. gingivalis. Analysis ofP. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment ofP. gingivaliswith protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production inP. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with aP. gingivalismutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with theP. gingivaliswild-type strain. These results indicate thatP. gingivaliscan temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.
Cytokine and Chemokine Production by Human Oral and Vaginal Epithelial Cells in Response toCandida albicans
Authors: Steele Chad , Fidel Paul L. 
Published: July 2002

ABSTRACTOropharyngeal and vaginal candidiases are the most common forms of mucosal fungal infections and are primarily caused byCandida albicans, a dimorphic fungal commensal organism of the gastrointestinal and lower female reproductive tracts. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral and vaginal mucosa duringC. albicansinfections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and sinceC. albicansis closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines and/or chemokines produced by primary oral and vaginal epithelial cells and cell lines in response toC. albicans. The results showed that proinflammatory cytokines were produced by oral and/or vaginal epithelial cells at various levels constitutively with considerable interleukin-1α (IL-1α) and tumor necrosis factor alpha, but not IL-6, produced in response toC. albicans. In contrast, Th1-type (IL-12 and gamma interferon) and Th2-type-immunoregulatory (IL-10 and transforming growth factor β) cytokines and the chemokines monocyte chemoattractant protein 1 and IL-8 were produced in low to undetectable concentrations with little additional production in response toC. albicans. Taken together, these results indicate that cytokines and chemokines are variably produced by oral and vaginal epithelial cells constitutively, as well as in response toC. albicans, and are predominated by proinflammatory cytokines.
Identification by Mass Spectrometry of CD8+-T-CellMycobacterium tuberculosisEpitopes within the Rv0341 Gene Product
Authors: Flyer David C. , Ramakrishna Venkatesh , Miller Cara , Myers Helen , McDaniel Melanie , Root Karen , Flournoy Caroline , Engelhard Victor H. , Canaday David H. , Marto Jarrod A. , Ross Mark M. , Hunt Donald F. , Shabanowitz Jeffrey , White Forest M. 
Published: July 2002

ABSTRACTIdentification ofMycobacterium tuberculosisproteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes fromM. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a singleM. tuberculosisprotein that is encoded by theM. tuberculosisRv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8+T cells from healthy blood donors in vitro and capable of recognizing and lysingM. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification ofM. tuberculosisproteins that can be evaluated as potential vaccine candidates.
Bacteria Induce Osteoclastogenesis via an Osteoblast-Independent Pathway
Authors: Jiang Yanling , Mehta Chetan K. , Hsu Tun-Yi , Alsulaimani Fahad F. H. 
Published: July 2002

ABSTRACTBacteria or their products may cause chronic inflammation and subsequent bone loss. This inflammation and bone loss may be associated with significant morbidity in chronic otitis media, periodontitis, endodontic lesions, and loosening of orthopedic implants caused by lipopolysaccharide (LPS)-contaminated implant particles. Currently, it is not clear how bacteria or endotoxin-induced bone resorption occurs and what cell types are involved. Here we report thatPorphyromonas gingivalis, a periodontal pathogen, andEscherichia coliLPS induce osteoclastic cell formation from murine leukocytes in the absence of osteoblasts. In contrast, stimulation with parathyroid hormone had no effect. These multinucleated, tartrate-resistant acid phosphatase-positive cells were positive for receptor activator of NF-κB (RANK), the receptor for osteoprotegerin ligand (OPGL), also known as RANK ligand (RANKL). Blocking antibodies demonstrated that their formation was dependent upon expression of OPGL and, to a lesser extent, on tumor necrosis factor alpha. Mononuclear cells represented a significant source of OPGL production. In vivo,P. gingivalisinjection stimulated OPGL expression in both mononuclear leukocytes and osteoblastic cells. Thus, these findings describe a pathway by which bacteria could enhance osteolysis independently of osteoblasts and suggest that the mix of cells that participate in inflammatory and physiologic bone resorption may be different. This may give insight into new targets of therapeutic intervention.
A Parasitic Phase-Specific Adhesin of
Coccidioides immitis
Contributes to the Virulence of This Respiratory Fungal Pathogen
Authors: Hung Chiung-Yu , Yu Jieh-Juen , Seshan Kalpathi R. , Reichard Utz , Cole Garry T. 
Published: July 2002

ABSTRACT

We report the isolation of a
Coccidioides immitis
gene (
SOWgp
) which encodes an immunodominant, spherule outer wall glycoprotein that is presented as a component of a parasitic phase-specific, membranous layer at the cell surface. The open reading frame of the gene from
C. immitis
isolate C735 translates a 422-amino-acid (aa) polypeptide that contains 6 copies of a 41- to 47-residue tandem repeat enriched in proline (20.4 mol%) and aspartate (19.7%). Two additional isolates of
C. immitis
produce SOWgps of different molecular sizes (328 and 375 aa) and show a corresponding difference in the number of tandem repeats (four and five, respectively). The accurate molecular sizes of these proline-rich antigens, as determined by surface-enhanced laser desorption/ionization mass spectrometry, are comparable to the predicted sizes from the translated protein sequences rather than the estimated sizes based on gel-electrophoretic separation. The results of Northern hybridization confirmed that
SOWgp
expression is parasitic phase specific, and immunoblot studies showed that elevated levels of production of this antigen occurred during early spherule development. The recombinant polypeptide (rSOWp) was shown to bind to mammalian extracellular matrix (ECM) proteins in an in vitro assay (laminin > fibronectin > collagen type IV), suggesting that the parasitic cell surface antigen may function as an adhesin. Deletion of the
SOWgp
gene by using a targeted gene replacement strategy resulted in partial loss of the ability of intact spherules to bind to ECM proteins and a significant reduction in virulence of the mutant strain. The wild-type gene was restored in the mutant by homologous recombination, and the revertant strain was shown to be as virulent as the parental isolate in our murine model of coccidioidomycosis. The parasitic cell surface glycoprotein encoded by the
SOWgp
gene appears to function as an adhesin and contributes to the virulence of
C. immitis.
Streptococcus-Zebrafish Model of Bacterial Pathogenesis
Authors: Neely Melody N. , Pfeifer John D. , Caparon Michael 
Published: July 2002

ABSTRACTDue to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish,Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that ofS. iniaein farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogenStreptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.
Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate
Authors: Stafslien D. K. , Cleary P. P. 
Published: July 2002

ABSTRACT

A glutathione-
S
-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca
2+
, Mg
2+
, and Mn
2+
but was inhibited by the same concentrations of Zn
2+
. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp
130
-Ala, His
193
-Ala, Asn
295
-Ala, and Ser
512
-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of
Streptococcus pyogenes
(group A streptococci), and recombinant SCPB, from
Streptococcus agalactiae
(group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP
−1
liter
−1
min
−1
.
Dual Control of
sbo-alb
Operon Expression by the Spo0 and ResDE Systems of Signal Transduction under Anaerobic Conditions in
Bacillus subtilis
Authors: Nakano Michiko M. , Zheng Guolu , Zuber Peter 
Published: July 2002

ABSTRACT

The
Bacillus subtilis sbo-alb
operon contains
sboA
, the structural gene for the bacteriocin subtilosin, and the
alb
genes required for subtilosin production. Transcription from the
sbo-alb
promoter is highly induced by oxygen limitation. The transcriptional regulation of the
sbo-alb
operon is under dual control involving the transition state regulator AbrB and the two-component regulatory proteins ResD and ResE.
The N- and C-Terminal Portions of the
Agrobacterium
VirB1 Protein Independently Enhance Tumorigenesis
Authors: Llosa Matxalen , Zupan John , Baron Christian , Zambryski Patricia 
Published: July 2002

ABSTRACT

Genetic transformation of plants by
Agrobacterium tumefaciens
is mediated by a virulence (
vir
)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a
virB1
deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a
virB1
deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other
vir
genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the
virB1
deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to
vir
regulation.
Autodisplay: Functional Display of Active β-Lactamase on the Surface of
Escherichia coli
by the AIDA-I Autotransporter
Authors: Lattemann Claus T. , Maurer Jochen , Gerland Elke , Meyer Thomas F. 
Published: July 2002

ABSTRACT

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the
Escherichia coli
adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme β-lactamase, leading to efficient expression of the fusion protein in
E. coli
. The β-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located β-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of β-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of
E. coli
cells expressing the β-lactamase AIDA-I fusion protein remained physiologically intact.
Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile
Authors: Bera Aloke Kumar , Chen Sihong , Smith Janet L. , Zalkin Howard 
Published: July 2002

ABSTRACT

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from
Aquifex aeolicus
were expressed in
Escherichia coli
, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH
3
from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH
3
for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.
Role of the
Pseudomonas aeruginosa oxyR-recG
Operon in Oxidative Stress Defense and DNA Repair: OxyR-Dependent Regulation of
katB-ankB
,
ahpB
, and
ahpC-ahpF
Authors: Ochsner Urs A. , Vasil Michael L. , Alsabbagh Eyad , Parvatiyar Kislay , Hassett Daniel J. 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
possesses an extensive armament of genes involved in oxidative stress defense, including
katB-ankB
,
ahpB
, and
ahpC-ahpF
. Transcription of these genes was regulated in response to H
2
O
2
, paraquat, or organic peroxides. Expression of
katB-lacZ
and the observed KatB catalase levels in
P. aeruginosa
PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also,
ahpB-lacZ
and
ahpC-lacZ
expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 μM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a Δ
oxyR
mutant, indicating that OxyR was essential for this response. The transcriptional start sites of
katB-ankB
,
ahpB
, and
ahpC-ahpF
were mapped, putative OxyR-binding sites were identified upstream of the −35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The
oxyR
mutant was hypersusceptible to oxidative stress-generating agents, including H
2
O
2
and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The
oxyR
phenotype was fully complemented by a plasmid containing the
oxyR
gene, while any of the
katB
,
ahpB
, or
ahpCF
genes alone resulted in only marginal complementation. Increased
katB-lacZ
expression and higher KatB catalase levels were detected in a Δ
ahpCF
background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In
P. aeruginosa
,
oxyR
is located upstream of
recG
, encoding a putative DNA repair enzyme.
oxyR-lacZ
and
recG-lacZ
reporter activities and
oxyR-recG
mRNA analysis showed that
oxyR
and
recG
are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of
oxyR
. Mutants affected in
recG
but not
oxyR
were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the
oxyR-recG
locus is essential for oxidative stress defense and for DNA repair.
Antigen 43 from
Escherichia coli
Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of
Pseudomonas fluorescens
Authors: Kjærgaard Kristian , Schembri Mark A. , Hasman Henrik , Klemm Per 
Published: July 2002

ABSTRACT

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of
Escherichia coli
. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of
E. coli
cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in
E. coli
. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important
Pseudomonas fluorescens
strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between
E. coli
and
P. fluorescens
. Furthermore, Ag43 expression enhances biofilm formation in
P. fluorescens
to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.