Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin.
Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX(Vc)), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX(Vc) (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324-1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding.
Kudryashov
Dmitri S
DS
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095.
Cordero
Christina L
CL
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.
Reisler
Emil
E
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095.
Satchell
Karla J Fullner
KJF
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611. Electronic address: k-satchell@northwestern.edu.
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008840:Microfilament Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014734:Vibrio cholerae / Q000378:metabolism*
D000199:Actins; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D000255:Adenosine Triphosphate