Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.
Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.
Ciesielski-Treska
J
J
Unité INSERM U-338, Strasbourg, France.
Ulrich
G
G
Baldacini
O
O
Monteil
H
H
Aunis
D
D
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D016360:Clostridioides difficile / Q000737:chemistry; D003013:Clostridium / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism; D003603:Cytotoxins / Q000378:metabolism*; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D009024:Morphogenesis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D014335:Tropomyosin / Q000378:metabolism; D016596:Vinculin / Q000276:immunology / Q000302:isolation & purification
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D000939:Epitopes; D010750:Phosphoproteins; D014335:Tropomyosin; C031815:toxB protein, Clostridium difficile; C073548:toxin L, Clostridium sordellii; D016596:Vinculin; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C