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- Title
- THE STRUCTURAL AND FOLDING CHARACTERISTICS OF THE PLASMID-ENCODED TOXIN FROM ENTEROAGGREGATIVE ESCHERICHIA COLI.
- Creator
-
Scaglione, Patricia, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β...
Show morePlasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β domain then forms a β-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the β-barrel at the extracellular surface to release a soluble toxin. This is thought to be a self-contained process that does not require chaperones or ATP for folding and export of the passenger domain. Pet produces cytotoxic effects through cleavage of its target, the actin-binding protein α- fodrin. Pet is secreted into the extracellular environment, but its target lies within the cytosol. To reach its target, Pet moves from the cell surface to the ER where it triggers ER-associated degradation (ERAD) to enter the cytosol. ERAD is a normal cellular process in which improperly folded proteins are exported from the ER to the cytosol for degradation. Other toxins that utilize this pathway are AB toxins such as cholera toxin (CT) and ricin. The A subunits of these toxins are thermally unstable, and this facilitates their ERAD-dependent translocation into the cytosol. Pet, however, is not an AB toxin. We predict that thermal unfolding is not the mechanism Pet employs to exploit ERAD. It was necessary to purify the toxin first in order to study the structural properties and ER export of Pet. Surprisingly, purified Pet eluted as two close peaks by size exclusion chromatography. Both peaks were Pet as demonstrated through immunoblotting. The folding efficiency of autotransporters has not been extensively elucidated, and based on our purification results, we hypothesized that there is inefficiency in the folding of autotransporters, specifically Pet. A toxicity assay showed that Pet peak one did not display cytopathic activity while Pet peak two did. CD and fluorescence spectroscopy measurements also detected structural differences between the two variants of Pet and demonstrated that Pet peak one was an unfolded variant of Pet peak two. Native gel electrophoresis and biophysical measurements indicated that Pet peak one did not exist as a dimer or aggregate. Our results indicate there are two forms of Pet, and thus the folding process of autotransporters appears to be inherently inefficient. Active Pet (peak two) was used for further biophysical measurements and biochemical assays. Circular dichroism and fluorescence spectroscopy showed that the secondary and tertiary structures of Pet are maintained at physiological temperature, 37°C. Thermal unfolding of Pet occurred at temperatures above 50°C. Fluorescence quenching of Pet was also performed and demonstrated that, at 37°C, there are solvent-exposed aromatic amino acids. The slight structural alterations to Pet at physiological temperature as well as the exposed hydrophobic residues could trigger ERAD. In addition, a modeled structure of Pet revealed a hydrophobic loop which is surface-exposed and a likely target for toxin-ERAD interactions. The data suggests that translocation of Pet mediated by ERAD can occur by a mechanism different from certain AB toxins. An open, hydrophobic conformation likely triggers ERAD, but may also contribute to poor folding.
Show less - Date Issued
- 2008
- Identifier
- CFE0002399, ucf:47761
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002399
- Title
- Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients.
- Creator
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Alcedo, Karel, Naser, Saleh, Cheng, Zixi, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three...
Show moreCrohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs (-) Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
Show less - Date Issued
- 2015
- Identifier
- CFE0005917, ucf:50831
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005917
