bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sensitivity of Polarized Epithelial Cells to the Pore-Forming Toxin Aerolysin
Authors: Abrami Laurence , Fivaz Marc , Glauser Pierre-Etienne , Sugimoto Nakaba , Zurzolo Chiara , van der Goot F. Gisou 
Published: January 2003

ABSTRACT

Aerolysin is one of the major virulence factors produced by
Aeromonas hydrophila
, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin
Clostridium septicum
alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.
Saccharomyces boulardiiInterferes with EnterohemorrhagicEscherichia coli-Induced Signaling Pathways in T84 Cells
Authors: Dahan Stephanie , Dalmasso Guillaume , Imbert Veronique , Peyron Jean-Francois , Rampal Patrick , Czerucka Dorota 
Published: January 2003

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) infections are associated with the modification of tight-junction permeability and synthesis of proinflammatory cytokine interleukin-8 (IL-8). In a previous study, it was demonstrated that EHEC-induced IL-8 secretion is due to the involvement of the mitogen-activated protein kinase (MAPK), AP-1, and NF-κB pathways. In this study, we investigated the effect of the yeastSaccharomyces boulardiion EHEC infection in T84 cells. For this purpose, cells were (i) incubated with bacteria and yeast at the same time or (ii) incubated overnight with yeast cells that were maintained during infection or eliminated by several washes before infection. Coincubation is sufficient to maintain the transmonolayer electrical resistance (TER) of EHEC-infected cells, whereas the preincubation of cells with the yeast without elimination of the yeast during infection is necessary to significantly decrease IL-8 secretion. We thus analyzed the mechanisms ofS. boulardiiaction. We showed thatS. boulardiihas no effect on EHEC growth or on EHEC adhesion. Kinetics studies revealed that EHEC-induced myosin light chain (MLC) phosphorylation precedes the decrease of TER. ML-7, an MLC kinase inhibitor, abolishes the EHEC-induced MLC phosphorylation and decrease of TER. Studies show thatS. boulardiialso abolishes EHEC-induced MLC phosphorylation. We demonstrated that the preincubation of cells withS. boulardiiwithout washes before EHEC infection inhibits NF-κB DNA binding activity, phosphorylation and degradation of IκB-α, and activation of the three members of a MAPK group (extracellular signal-regulated protein kinases 1 and 2, p38, and c-jun N-terminal kinase). These findings demonstrate thatS. boulardiiexerts a preventive effect on EHEC infection by (i) interfering with one of the transduction pathways implicated in the control of tight-junction structure and (ii) decreasing IL-8 proinflammatory secretion via inhibition of the NF-κB and MAPK signaling pathways in infected T84 cells.
Streptococcus pyogenesGlycoprotein-Binding Strepadhesin Activity Is Mediated by a Surface-Associated Carbohydrate-Degrading Enzyme, Pullulanase
Authors: Hytönen Jukka , Haataja Sauli , Finne Jukka 
Published: January 2003

ABSTRACTThe interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity inStreptococcus pyogenesbinding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in anS. pyogenesstrain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of thepulAgene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus,S. pyogenesstrepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.
Five-Member Gene Family of
Bartonella quintana
Authors: Minnick Michael F. , Sappington Kate N. , Smitherman Laura S. , Andersson Siv G. E. , Karlberg Olof , Carroll James A. 
Published: January 2003

ABSTRACT

Bartonella quintana
, the agent of trench fever and an etiologic agent of bacillary angiomatosis, has an extraordinarily high hemin requirement for growth compared to other bacterial pathogens. We previously identified the major hemin receptor of the pathogen as a 30-kDa surface protein, termed HbpA. This report describes four additional homologues that share approximately 48% amino acid sequence identity with
hbpA
. Three of the genes form a paralagous cluster, termed
hbpCAB
, whereas the other members,
hbpD
and
hbpE
, are unlinked. Secondary structure predictions and other evidence suggest that Hbp family members are β-barrels located in the outer membrane and contain eight transmembrane domains plus four extracellular loops. Homologs from a variety of gram-negative pathogens were identified, including
Bartonella henselae
Pap31,
Brucella
Omp31,
Agrobacterium tumefaciens
Omp25, and neisserial opacity proteins (Opa). Family members expressed in vitro-synthesized proteins ranging from ca. 26.5 to 35.1 kDa, with the exception of HbpB, an ∼55.9-kDa protein whose respective gene has been disrupted by a ∼510 GC-rich element containing variable-number tandem repeats. Transcription analysis by quantitative reverse transcriptase-PCR (RT-PCR) indicates that all family members are expressed under normal culture conditions, with
hbpD
and
hbpB
transcripts being the most abundant and the rarest, respectively. Mutagenesis of
hbpA
by allelic exchange produced a strain that exhibited an enhanced hemin-binding phenotype relative to the parental strain, and analysis by quantitative RT-PCR showed elevated transcript levels for the other
hbp
family members, suggesting that compensatory expression occurs.
Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells
Authors: Obrig T. G. , Seaner R. M. , Bentz M. , Lingwood C. A. , Boyd B. , Smith A. , Narrow W. 
Published: January 2003

ABSTRACT

Shiga toxin-producing enterohemorrhagic
Escherichia coli
is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.
Protection of Chickens againstEscherichia coliInfections by DNA Containing CpG Motifs
Authors: Gomis Susantha , Babiuk Lorne , Godson Dale L. , Allan Brenda , Thrush Tannis , Townsend Hugh , Willson Philip , Waters Edwin , Hecker Rolf , Potter Andrew 
Published: January 2003

ABSTRACTSynthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate ofEscherichia coliwas applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after theE. colichallenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN(2007)had a survival rate of 15%. In contrast, groups that received CpG-ODN(2007), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P< 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN(2007)by the subcutaneous route (P< 0.01). A dose of as little as 3.16 μg of CpG-ODN(2007), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds againstE. coliinfection (P< 0.01). This study demonstrates that CpG-ODN(2007)has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN(2007)has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.
Disruption of Epithelial Barrier Integrity bySalmonella entericaSerovar Typhimurium Requires Geranylgeranylated Proteins
Authors: Tafazoli Farideh , Magnusson Karl-Eric , Zheng Limin 
Published: January 2003

ABSTRACTEpithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such asSalmonellaandYersiniaspp., have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasiveSalmonella entericaserovar Typhimurium SL1344 exhibited marked changes in F-actin organization, an increase in the paracellular flux of dextran, and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with an isogenic noninvasive mutant (hilA) increased the TER in these cells. Pretreating MDCK-1 cells with the inhibitors for tyrosine kinase (genistein) or phosphatidylinositol 3-kinase (wortmannin) did not affect invasion and subsequent perturbation of the epithelial barrier by serovar Typhimurium. Instead, the geranylgeranyltransferase 1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of serovar Typhimurium to disrupt the epithelial barrier. The substrates for GGTI-298 include Rho family GTPases, as indicated by inhibiting prenylation of Rac1 and Cdc42. Infection with wild-type serovar Typhimurium increased the level of activated Rac1 and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. ThisSalmonella-induced accumulation of Rac1 and Cdc42 and alteration of the junction-associated proteins ZO-1, occludin, and E-cadherin in MDCK-1 cells were markedly inhibited by GGTI-298. These results suggest that activation of geranylgeranylated proteins, including Rac1 and Cdc42, is critical for disruption of barrier integrity by serovar Typhimurium in polarized MDCK-1 cells.
Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model
Authors: Kadurugamuwa Jagath L. , Sin Lin , Albert Eddie , Yu Jun , Francis Kevin , DeBoer Monica , Rubin Michael , Bellinger-Kawahara Carole , Parr, T. R. , Contag Pamela R. 
Published: January 2003

ABSTRACT

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens,
Staphylococcus aureus
and
Pseudomonas aeruginosa
, were made bioluminescent by insertion of a complete
lux
operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (
S. aureus r
= 0.98;
P. aeruginosa r
= 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10
3
to 10
5
CFU/catheter for
S. aureus
and
P. aeruginosa
resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the
lux
constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.
Colonial Morphology of
Burkholderia cepacia
Complex Genomovar III: Implications in Exopolysaccharide Production, Pilus Expression, and Persistence in the Mouse
Authors: Chung Jacqueline W. , Altman Eleonora , Beveridge Terry J. , Speert David P. 
Published: January 2003

ABSTRACT

The purpose of this study was to determine the role of colonial morphology of
Burkholderia cepacia
complex (BCC) organisms in pathogenicity in a mouse model of pulmonary infection. BCC strain C1394 was rapidly cleared by leukopenic mice after intranasal challenge, whereas a spontaneous variant (C1394mp2) that was indistinguishable from the parent strain by genetic typing persisted in the lungs and differed in colonial morphology. The parent strain had a matte colonial phenotype, made scant exopolysaccharide (EPS), and was lightly piliated. The variant had a shiny phenotype, produced abundant EPS, and was heavily piliated. Matte to shiny colonial transformation was induced by growth at 42°C. Colonial morphology in the BCC strain variant was associated with persistence after pulmonary challenge and appeared to be correlated with the elaboration of putative virulence determinants.
Vaccine-Induced Reduction ofHelicobacter pyloriColonization in Mice Is Interleukin-12 Dependent but Gamma Interferon and Inducible Nitric Oxide Synthase Independent
Authors: Garhart Christine A. , Heinzel Frederick P. , Czinn Steven J. , Nedrud John G. 
Published: January 2003

ABSTRACTPrevious studies with mice have shown that major histocompatibility complex class II (MHC-II) is required for protection fromHelicobacter pylori, while MHC-I and antibodies are not. Thus, CD4+T cells are presumed to play an essential role in protective immunity via secretion of cytokines. To determine which cytokines are associated with a reduction of bacterial load in immunized mice, gastric cytokine expression was examined by semiquantitative reverse transcription-PCR in protected (defined as ≥2-log-unit decrease in bacterial load) and unprotected mice 4 weeks after challenge. Elevated levels of mRNA for interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) were associated with protection in immunized-challenged (I/C) mice, but Th2 cytokine (IL-4, IL-5, IL-10, and IL-13) and chemokine (KC, MIP-2, and MCP-1) expression was not associated with protection. Despite the association of IFN-γ and iNOS message with protection, I/C mice genetically lacking either of these products were able to reduce the bacterial load as well as the wild-type I/C controls. The I/C mice lacking IL-12p40 were not protected compared to unimmunized-challenged mice. All I/C groups developed gastritis. We conclude that neither IFN-γ nor iNOS is essential for vaccine-induced protection fromH. pyloriinfection. The p40 subunit of IL-12, which is a component of both IL-12 and IL-23, is necessary for protection in immunized mice. These findings suggest a novel IFN-γ-independent function of IL-12p40 in effective mucosal immunization againstH. pylori.
Phagocytic Activity and Monocyte Chemotactic Protein Expression by Pulmonary Macrophages in Persistent Pulmonary Cryptococcosis
Authors: He Wu , Casadevall Arturo , Lee Sunhee C. , Goldman David L. 
Published: January 2003

ABSTRACTThe mechanisms by whichCryptococcus neoformanspersists in an immunocompetent host are not well understood. Using a rat model of persistent infection, we investigated the ability of pulmonary macrophages (PuM) to phagocytizeC. neoformansand produce monocyte chemotactic protein 1 (MCP-1) as a function of the length of time of infection and opsonin. The ability of macrophages to affect serum-mediated phagocytosis varied over the course of infection and was dependent on CD11b/c and CD18 expression. Infection resulted in increased MCP-1 levels within the lung, though the actual amounts varied over the course of infection. Immunohistochemical studies localized MCP-1 expression to macrophages and epithelioid cells. Enhanced production of MCP-1 by PuM from infected rats was confirmed by ex vivo studies. Induction of MCP-1 following serum-mediated phagocytosis was observed for PuM from both infected and noninfected rats and depended on the interaction ofC. neoformanswith CD11b/c and CD18. Specific antibody was more efficient than serum in promoting phagocytosis and consistently elicited more MCP-1. The relative amount of MCP-1 produced in association with phagocytosis was similar for PuM at all lengths of time of infection. Decreased MCP-1 production was observed for PuM obtained from older rats, including long-term (8 to 10 months)-infected and age-matched controls, suggesting that aging may affect the production of MCP-1 by PuM in response to cryptococcal infection. In summary, our results show that macrophages are an important source of MCP-1 during pulmonary cryptococcosis and that MCP-1 production is actively regulated during infection. Furthermore, we find that phagocytosis ofC. neoformanscan serve as an important stimulus for MCP-1 production by PuM, though the efficiency of this process is dependent on the opsonin type and may be affected by aging.
Infection byStreptococcus pyogenesInduces the Receptor Activator of NF-κB Ligand Expression in Mouse Osteoblastic Cells
Authors: Okahashi Nobuo , Sakurai Atsuo , Nakagawa Ichiro , Fujiwara Taku , Kawabata Shigetada , Amano Atsuo , Hamada Shigeyuki 
Published: January 2003

ABSTRACTGroup AStreptococcus pyogenesis known to induce nongonococcal septic arthritis in addition to pharyngitis, scarlet fever, and poststreptococcal sequelae. However, little is known about the interaction betweenS. pyogenesand bone cells. We report here thatS. pyogenesstrain JRS4 (M6) attached to and invaded mouse primary osteoblasts. Reverse transcription-PCR demonstrated thatS. pyogenesinfection of osteoblasts stimulated expression of mRNA for the receptor activator of NF-κB ligand (RANKL). Western blot analysis followed by ligand precipitation with the receptor activator of NF-κB receptor showed that there was an increase in RANKL protein in infected osteoblasts. Production of interleukin-6 was also stimulated, but no production of interleukin-1β or tumor necrosis factor alpha was observed. Stimulation of RANKL production was not observed in osteoblasts stimulated with heat-inactivatedS. pyogenes, suggesting that an active interaction ofS. pyogeneswith osteoblasts is essential for this phenomenon. A Western blot analysis performed with antibodies specific for phosphorylated signal transduction proteins demonstrated thatS. pyogenesinfection induces phosphorylation of p38 mitogen-activated protein kinase. A specific inhibitor of this kinase, SB203580, inhibited RANKL production by infected osteoblasts. These results suggest that infection of osteoblasts byS. pyogenesstimulates RANKL production and may trigger bone destruction in infected bone tissue.
Immunohistochemical Evaluation of T Cells in Oral Lesions from Human Immunodeficiency Virus-Positive Persons with Oropharyngeal Candidiasis
Authors: Myers Tammy A. , Leigh Janet E. , Arribas Alfredo R. , Hager Shannon , Clark Rebecca , Lilly Elizabeth , Fidel, Paul L. 
Published: January 2003

ABSTRACTOropharyngeal candidiasis (OPC), caused byCandida albicans, is the most frequent opportunistic fungal infection in human immunodeficiency virus (HIV)-positive persons. Although Th1-type CD4+T cells are considered important for host defense against mucosalC. albicansinfections, there is a paucity of information regarding the presence and/or role of T cells in OPC lesions. In pursuit of this, initial chromophore immunohistochemical studies showed a majority of CD8+rather than CD4+cells equally distributed throughout the buccal mucosa of OPC−persons (HIV−or HIV+), irrespective of blood CD4+cell numbers. In contrast, CD8+cells in lesions from HIV+OPC+persons were in significantly higher numbers and concentrated at the lamina propria-epithelium interface, a considerable distance from theCandidaat the outer epithelium. Dual fluorescence and confocal microscopy confirmed that the majority of CD8+, but not CD4+, cells were T cells by the presence or absence, respectively, of CD3 on each cell type. These results suggest that CD8+T cells may be important for oral host defense against OPC, especially when CD4 cell numbers are reduced, with a potential CD8 cell-specific dysfunction associated with susceptibility to OPC.