Abstract
Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg2+-GTP to support crosslinking, but the kinetic parameters (KM = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.
Highlights
Actin is a ubiquitous and highly conserved eukaryotic protein intrinsically involved in numerous vital processes in living cells
We established the mechanism by which the energy of ATP is utilized for actin crosslinking by the V. cholerae Actin Crosslinking Domain (ACD) toxin and determined the kinetic parameters and pH optimum for this reaction
Our findings provide evidence that ACD utilizes the energy of Mg2+-ATP/GTP for activation of the E270 residue of actin
Summary
Actin is a ubiquitous and highly conserved eukaryotic protein intrinsically involved in numerous vital processes in living cells. Depending on the carrier toxin, ACD can be delivered to the cytoplasm of the host cell via either Type I (MARTX) [5,6] or Type VI (VgrG1) [2] secretion systems. Both ACD containing toxins of V. cholerae contribute to inflammatory diarrhea, prolonged colonization of the small intestine and lethality in experimental mouse models [7,8,9,10], suggesting that ACD has generally evolved as a factor compromising the host’s immune system
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