NDLI: Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity

Content Provider	PubMed Central
Author	Suhan, Michelle L. Hovde, Carolyn J.
Editor	Barbieri, J. T.
Copyright Year	1998
Abstract	Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.
Starting Page	5252
File Format	PDF
ISSN	10985522
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	11
Volume Number	66
Language	English
Publisher	American Society for Microbiology
Publisher Date	1998-11-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1

Shiga toxin-associated hemolytic uremic syndrome: interleukin-1 beta enhancement of Shiga toxin cytotoxicity toward human vascular endothelial cells in vitro.

Shiga Toxin 1 Causes Direct Renal Injury in Rats

Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and α-Sarcin▿

Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography.

Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits

Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1▿ †

Bcl-2 Regulates the Onset of Shiga Toxin 1-Induced Apoptosis in THP-1 Cells ▿

Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity

Similar Documents

Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1

Shiga toxin-associated hemolytic uremic syndrome: interleukin-1 beta enhancement of Shiga toxin cytotoxicity toward human vascular endothelial cells in vitro.

Shiga Toxin 1 Causes Direct Renal Injury in Rats

Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and α-Sarcin▿

Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography.

Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits

Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1▿ †

Bcl-2 Regulates the Onset of Shiga Toxin 1-Induced Apoptosis in THP-1 Cells ▿

Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity