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Structural and signaling aspects of ...

Karve, Sayali.

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Structural and signaling aspects of Shiga toxin.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Structural and signaling aspects of Shiga toxin./
Author:	Karve, Sayali.
Description:	145 p.
Notes:	Source: Dissertation Abstracts International, Volume: 77-05(E), Section: B.
Contained By:	Dissertation Abstracts International77-05B(E).
Subject:	Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3741639
ISBN:	9781339339993

Structural and signaling aspects of Shiga toxin.
Karve, Sayali.

Structural and signaling aspects of Shiga toxin. - 145 p.

Source: Dissertation Abstracts International, Volume: 77-05(E), Section: B.

Thesis (Ph.D.)--University of Cincinnati, 2015.

Shiga toxin (Stx) producing E. coli (STEC) is a major cause of foodborne illnesses. Approximately 265,000 cases of STEC infections are reported annually in the United States, of which &sim;10% lead to the life-threatening complication, hemolytic uremic syndrome (HUS). The major virulence factor of STEC is Stx, an AB5 toxin that consists of a single A-subunit with ribosomal RNA-cleaving activity, surrounded by a receptor-binding B-pentamer. Two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to be important. Previous studies reported binding of Stx1 and Stx2a toxoids to glycolipid receptors. We studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all holotoxins significantly increased in presence of PC and Ch. Stx2a has been previously shown to form a less stable B-pentamer compared to Stx1. However, its effect on receptor binding is unknown. We showed that the more stable B-pentamer of Stx1 bound better to glycolipids than the less stable B-pentamer of Stx2a. However, B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for glycolipid binding of Stx1 than Stx2a.

ISBN: 9781339339993Subjects--Topical Terms:

536250
Microbiology.

Structural and signaling aspects of Shiga toxin.
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020 $a 9781339339993
035 $a (MiAaPQ)AAI3741639
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100 1 $a Karve, Sayali. $3 3183056
245 1 0 $a Structural and signaling aspects of Shiga toxin.
300 $a 145 p.
500 $a Source: Dissertation Abstracts International, Volume: 77-05(E), Section: B.
500 $a Adviser: Alison Weiss.
502 $a Thesis (Ph.D.)--University of Cincinnati, 2015.
520 $a Shiga toxin (Stx) producing E. coli (STEC) is a major cause of foodborne illnesses. Approximately 265,000 cases of STEC infections are reported annually in the United States, of which &sim;10% lead to the life-threatening complication, hemolytic uremic syndrome (HUS). The major virulence factor of STEC is Stx, an AB5 toxin that consists of a single A-subunit with ribosomal RNA-cleaving activity, surrounded by a receptor-binding B-pentamer. Two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to be important. Previous studies reported binding of Stx1 and Stx2a toxoids to glycolipid receptors. We studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all holotoxins significantly increased in presence of PC and Ch. Stx2a has been previously shown to form a less stable B-pentamer compared to Stx1. However, its effect on receptor binding is unknown. We showed that the more stable B-pentamer of Stx1 bound better to glycolipids than the less stable B-pentamer of Stx2a. However, B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for glycolipid binding of Stx1 than Stx2a.
520 $a STEC colonize the gastrointestinal tract and produce Stx. The mechanism by which Stx crosses the intestinal epithelial barrier to reach systemic circulation is unknown, since until recently no animal or cellular models were available that closely resembled the human intestine. We investigated stem cell derived 'induced human intestinal organoids' (iHIO), which maintain the human intestinal architecture and function, to study the intestinal actions of Stx and STEC. Purified Stx2a and Stx2a producing E. coli were microinjected into iHIO lumens. As negative controls, phosphate buffered saline (PBS), inactivated Stx2a toxoid, and non-pathogenic ECOR13 strain of E. coli were microinjected. iHIO treated with PBS and Stx2a toxoid remained intact; however, Stx2a caused loss of iHIO epithelial barrier function. Cryosections of PBS treated iHIO displayed well-defined lumens and intercellular E-cadherin, whereas luminal extrusion of cells and altered E-cadherin localization was observed for Stx2a treated iHIO. iHIO supported rapid growth of E. coli. Intact iHIO were observed after infection with ECOR13, whereas STEC caused a very rapid and severe damage, resulting in loss of 3-dimensional arrangement of iHIO. In case of ECOR13, cryosections revealed short rods of E. coli in lumens, surrounded by a well-defined epithelial layer with intercellular E-cadherin and apical F-actin. Conversely, STEC treated iHIO displayed cryosections filled with filamentous E. coli, damaged epithelial cells displaying mislocalized E-cadherin and F-actin. Overall these results suggest that iHIO are sensitive to Stx and STEC, and thus represent the first humanized model to study intestinal interaction of STEC.
590 $a School code: 0045.
650 4 $a Microbiology. $3 536250
650 4 $a Toxicology. $3 556884
690 $a 0410
690 $a 0383
710 2 $a University of Cincinnati. $b Medicine: Molecular Genetics, Biochemistry, and Microbiology. $3 3183057
773 0 $t Dissertation Abstracts International $g 77-05B(E).
790 $a 0045
791 $a Ph.D.
792 $a 2015
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3741639