scholarly journals The Aggregation Domain of Aggregation Substance, Not the RGD Motifs, Is Critical for Efficient Internalization by HT-29 Enterocytes

2003 ◽  
Vol 71 (10) ◽  
pp. 5682-5689 ◽  
Author(s):  
Christopher M. Waters ◽  
Carol L. Wells ◽  
Gary M. Dunny
Keyword(s):  
Enterococcus Faecalis ◽  
Surface Protein ◽  
Cell Types ◽  
Functional Domain ◽  
Aggregation Substance ◽  
Bacterial Aggregation ◽  
Bacterial Aggregates ◽  
Different Cell Types ◽  
Ht 29 ◽  
Dependent Mechanism

ABSTRACT Aggregation substance (AS), a surface protein encoded on the pheromone-inducible plasmids of Enterococcus faecalis, has been shown to increase adherence and internalization into a number of different cell types, presumably through integrin binding mediated by the N-terminal RGD motif of AS. Here, defined mutations constructed in Asc10, the AS encoded by the plasmid pCF10, are analyzed for their ability to promote increased internalization levels into HT-29 enterocytes. The results clearly show that the previously identified Asc10 functional domain, not the RGD motifs, is critical for Asc10-directed internalization of E. faecalis into HT-29 enterocytes. Also, expression of Asc10 in the nonaggregating E. faecalis strain INY3000 is unable to mediate HT-29 internalization. However, Asc10-expressing E. faecalis cells are not internalized as bacterial aggregates, suggesting bacterial aggregation is not a prerequisite for HT-29 internalization. These data show that Asc10 directs internalization of E. faecalis into HT-29 enterocytes through a non-RGD-dependent mechanism.

scholarly journals Inducible Expression of Enterococcus faecalis Aggregation Substance Surface Protein Facilitates Bacterial Internalization by Cultured Enterocytes

2000 ◽  
Vol 68 (12) ◽  
pp. 7190-7194 ◽  
Author(s):  
Carol L. Wells ◽  
Elizabeth A. Moore ◽  
Julie A. Hoag ◽  
Helmut Hirt ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Enterococcus Faecalis ◽  
Surface Protein ◽  
Inducible Expression ◽  
Free Medium ◽  
Heterologous Host ◽  
Aggregation Substance ◽  
Ht 29

ABSTRACT Aggregation substance (AS) is an Enterococcus faecalissurface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalisinternalization by cultured enterocytes.

scholarly journals Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis

2001 ◽  
Vol 69 (5) ◽  
pp. 3305-3314 ◽  
Author(s):  
John K. McCormick ◽  
Helmut Hirt ◽  
Christopher M. Waters ◽  
Timothy J. Tripp ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Enterococcus Faecalis ◽  
Rabbit Model ◽  
Surface Protein ◽  
Conjugative Plasmid ◽  
Aggregation Substance ◽  
Exposed Region ◽  
Correct Sequence

ABSTRACT The aggregation substance (AS) surface protein fromEnterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS44–331) was cloned with a C-terminal histidine tag translational fusion and expressed fromEscherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS44–331 reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS44–331 bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS44–331were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS44–331 were challenged intravenously withE. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due toE. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

scholarly journals Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy)

2003 ◽  
Vol 52 (6) ◽  
pp. 491-498 ◽  
Author(s):  
I. Duprè ◽  
S. Zanetti ◽  
A. M. Schito ◽  
G. Fadda ◽  
L. A. Sechi
Keyword(s):  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Virulence Genes ◽  
Protein Gene ◽  
Surface Protein ◽  
Virulence Determinants ◽  
Colonization Factor ◽  
Aggregation Substance ◽  
Epithelial Cell Lines ◽  
Surface Protein Gene

Enterococci are widely distributed in the environment; within the human body, they are normal commensals of the oral cavity, gastrointestinal tract and vagina. In recent years, enterococci have become one of the most frequent causes of acquired nosocomial infections worldwide. The molecular mechanism of virulence of these bacteria is still not completely understood. The aims of this work were to characterize phenotypically 47 isolates of Enterococcus faecalis and Enterococcus faecium collected in Sardinia (Italy) by their abilities to adhere to different epithelial cell lines (Vero and Caco-2 cells) and to associate their phenotypes with the presence of known virulence genes detected within their genomes by PCR. The following genes were amplified: AS (aggregation substance), esp (surface protein gene), ace (accessory colonization factor), efaA (E. faecalis endocarditis antigen) and gelE (gelatinase). The virulence genes were detected in E. faecalis isolates only, with the exception of esp, which was found in both species. The phenotypic and genotypic results were also compared with the susceptibility of isolates to various antibiotics.

scholarly journals Adhesion to Bile Drain Materials and Physicochemical Surface Properties of Enterococcus faecalis Strains Grown in the Presence of Bile

2002 ◽  
Vol 68 (8) ◽  
pp. 3855-3858 ◽  
Author(s):  
Karola Waar ◽  
Henny C. van der Mei ◽  
Hermie J. M. Harmsen ◽  
John E. Degener ◽  
Henk J. Busscher
Keyword(s):  
Surface Properties ◽  
Enterococcus Faecalis ◽  
Photoelectron Spectroscopy ◽  
Surface Protein ◽  
Lower Surface ◽  
Contact Angles ◽  
Surface Proteins ◽  
Water Contact ◽  
Zeta Potentials ◽  
Aggregation Substance

ABSTRACT The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.

Sustained hyposmotic stress induces cell death: apoptosis by defeat

2001 ◽  
Vol 281 (5) ◽  
pp. C1716-C1726 ◽  
Author(s):  
Tina Jäckle ◽  
Cornelia Hasel ◽  
Ingo Melzner ◽  
S. Brüderlein ◽  
Peter M. Jehle ◽  
...  
Keyword(s):  
Human Kidney ◽  
Cell Types ◽  
Surface Expression ◽  
Carcinoma Cells ◽  
Mitochondrial Mass ◽  
Hyposmotic Stress ◽  
Stressed Cells ◽  
Induced Apoptosis ◽  
Different Cell Types ◽  
Ht 29

We describe sustained hyposmotic stress as a novel type of environmental condition enforcing apoptosis. In a dose- and time-dependent fashion, hyposmotic stress leads to a delayed type of apoptosis with considerable variations in constitutive sensitivity among different cell types. For example, after 48 h at 84 mosmol/l, the death rate ranged from 10.8 ± 0.7% in AsPc1 human pancreatic carcinoma cells to 72.0 ± 1.6% in HK-2 human kidney tubule cells. Caspase inhibitors rendered cells more resistant to hyposmolar stress; the caspase 3 inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde was the most efficient. After 24 h of stress, HT-29 colon carcinoma and HK-2 cells had increased their mitochondrial mass. This went along with an increase in mitochondrial membrane potential in HT-29 cells but with a decrease in HK-2 cells. Starting at 2 h of stress, we detected transient CD95L transcription followed by surface expression of CD95L in HT-29 but not in HK-2 cells. Inhibitory CD95L antibody partially inhibited specific death in HT-29 but not in HK-2 cells. Thus, as in other types of stress-induced apoptosis, the CD95/CD95L system is one of the different routes to suicide optionally used by hyposmotically stressed cells. Our findings may have clinical implications for the prevention and treatment of tissue damage caused by severe hyposmolar states.

scholarly journals Effects of sarang semut (Myrmecodia Pendens Merr. & Perry) extracts on Enterococcus faecalis sensitivity

2016 ◽  
Vol 49 (4) ◽  
pp. 175
Author(s):  
Cut Soraya ◽  
Hendra Dian Adhyta Dharsono ◽  
Dudi Aripin ◽  
Mieke H Satari ◽  
Dikdik Kurnia ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Surface Protein ◽  
Host Cells ◽  
Minimal Bactericidal Concentration ◽  
Minimal Inhibition Concentration ◽  
Aggregation Substance ◽  
Inhibition Concentration ◽  
Low Sensitivity ◽  
Better Than ◽  
Positive Controls

Background: Enterococcus faecalis (E. faecalis) is a gram positive oral pathogen that reported at the main agent infection of endodontic treatment. Its activities are influenced by the virulence factors facilitating the interaction process between agents with host cells. Like aggregation substance, cytolysin, extracellular superoxide, gelatinase, hyaluronidase, sex pheromones, and surface adhesions molecules. Plant extracts are reported as the material antibacterial as well as E. faecalis in pathogenesis of endodontic infections. Purpose: Purpose of this study was to analyse of sarang semut extracts (Myrmecodia Pendens Merr. & Perry) towards sensitivity of E. faecalis. Method: This research used the methanol extract of sarang semut, E. faecalis ATCC 29212, and fosfomycin also chlorhexidine as the positive controls. Whereas, Bradford protein method was measured the concentration of the surface protein of E. faecalis and active component of the sarang semut extract. Result: Generally, the sarang semut extract possessed low sensitivity toward E. faecalis (≤ 13 mm), but on the concentrations of 100 µg/ml and 75 µg/ml better than inhibition of other concentrations, round 10.6-11.6 (mm). Specifically, on 100 µg/ml has indicator the minimal bactericidal concentration (MBC) on E. faecalis. Whereas minimal inhibition concentration (MIC) on the concentration of 3,125 µg/ml. Conclusion: Based on MBC and MIC assay, the extract of sarang semut has potential effects to adherence growth of E. faecalis, mainly on the highest concentration 100 µg/ml also MIC on 3,125 µg/ ml.

scholarly journals Aggregation Substance Promotes Adherence, Phagocytosis, and Intracellular Survival of Enterococcus faecalis within Human Macrophages and Suppresses Respiratory Burst

2000 ◽  
Vol 68 (9) ◽  
pp. 4900-4906 ◽  
Author(s):  
Sigurd D. Süßmuth ◽  
Albrecht Muscholl-Silberhorn ◽  
Reinhard Wirth ◽  
Milorad Susa ◽  
Reinhard Marre ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Respiratory Burst ◽  
Superoxide Anion ◽  
Surface Protein ◽  
Human Monocyte ◽  
Human Macrophages ◽  
Aggregation Substance ◽  
Type 3 ◽  
Inhibition Studies ◽  
Macrophage Killing

ABSTRACT The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express β2 integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.

scholarly journals PKA compartmentalization links cAMP signaling and autophagy

2021 ◽  
Author(s):  
Francesca Grisan ◽  
Liliana F. Iannucci ◽  
Nicoletta C. Surdo ◽  
Andrea Gerbino ◽  
Sofia Zanin ◽  
...  
Keyword(s):  
Molecular Machines ◽  
Cell Types ◽  
Type I ◽  
Cell Type ◽  
Signaling Events ◽  
Membrane Bound ◽  
Different Cell Types ◽  
Ht 29 ◽  
Camp Elevation

AbstractAutophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.

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