Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1

Journal of Microbiological Methods
Uma S BabuRichard B Raybourne

Abstract

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to de...Continue Reading

References

Jul 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·S B CalderwoodJ J Mekalanos
Aug 1, 1997·Journal of Clinical Microbiology·K S KehlD W Acheson
Aug 26, 1998·Infection and Immunity·D W AchesonM K Waldor
Oct 20, 1998·Lancet·P S Mead, P M Griffin
Nov 30, 2000·Current Infectious Disease Reports·J L Jaeger, D W Acheson
Feb 13, 2003·Pediatric Nephrology : Journal of the International Pediatric Nephrology Association·Richard L SieglerFletcher B Taylor
Aug 6, 2003·Biochemistry·David G PinaValery L Shnyrov
Nov 26, 2003·FEMS Microbiology Letters·Rama P CherlaVernon L Tesh
Jan 18, 2005·Journal of Dairy Science·H S Hussein, T Sakuma
Feb 3, 2006·FEMS Microbiology Letters·Susan M HarringtonJames P Nataro
Apr 1, 2006·Seminars in Thrombosis and Hemostasis·L Bernd ZimmerhacklUNKNOWN European Study Group for Haemolytic Uraemic Syndromes and Related Disorders
Jun 8, 2006·Nephrology·Iradj Amirlak, Bardia Amirlak

❮ Previous
Next ❯

Citations

Mar 10, 2010·Future Microbiology·Vernon L Tesh
Oct 20, 2020·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Xinyuan ZhangKeyue Shen

❮ Previous
Next ❯

Related Concepts

Related Feeds

Bacteriophage: Phage Therapy

Phage therapy uses bacterial viruses (bacteriophages) to treat bacterial infections and is widely being recognized as an alternative to antibiotics. Here is the latest research.

Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis