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Fodrin CaM‐binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption
Author(s) -
CanizalezRoman Adrián,
NavarroGarcía Fernando
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03492.x
Subject(s) - spectrin , biology , cytoskeleton , actin , microbiology and biotechnology , calmodulin , calmodulin binding proteins , serine protease , protease , biochemistry , cell , enzyme
Summary We have previously shown that the plasmid‐encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet‐intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non‐erythroid spectrin). Here we demonstrate that Pet has affinity for α‐fodrin (formally named αII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST‐fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M 1198 and V 1199 , in the calmodulin binding domain. Site‐directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet‐treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST‐fodrin. This is the first report showing cleavage of α‐fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.