The Actin cross-linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin.
Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF(N)ACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF(N)ACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg(2+) and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.
Cordero
Christina L
CL
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, USA.
Kudryashov
Dmitry S
DS
Reisler
Emil
E
Satchell
Karla J Fullner
KJ
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D017434:Protein Structure, Tertiary; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity
D000199:Actins; D001426:Bacterial Proteins; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D000217:Acyltransferases; C434465:RtxC protein, Vibrio cholerae