Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans

Abstract

Homologous bacterial metalloproteases ECP32 ⁄ grimelysin from Serratia grimesii and protealysin from Serratia proteamaculans are involved in the invasion of the nonpathogenic bacteria in eukaryotic cells and are suggested to translocate into the cytoplasm [Bozhokina ES et al. (2011) Cell Biol Int 35, 111-118]. The proteases have been characterized as actinhydrolyzing enzymes with a narrow specificity toward intact cell proteins. However, cleavage of filamentous actin (F-actin) (i.e. the main actin species in the cell) and the properties of the cleaved F-actin have not been investigated previously. In the present study, we revealed the presence of protealysin in the cytoplasm of 3T3-SV40 cells infected with S. proteamaculans or recombinant Escherichia coli expressing the protealysin gene. We also show for the first time that purified protealysin and the lysates of the recombinant E. coli producing protealysin cleave 20-40% of F-actin. Cleavage limited predominantly to the bond Gly42-Val43 efficiently increases the steady-state ATPase activity (dynamics) of F-actin. AlF À 4 abolishes this effect and promotes the nucleation of protealysin-cleaved Mg-globularactin even in the absence of 0.1 M KCl, most likely as a result of the stabilization of lateral intermonomer contacts of actin subunits. The results obtained in the present study suggest that F-actin can be a target for protealysin upon its translocation into the host cell.

The Serratia grimesii outer membrane vesicles‐associated grimelysin triggers bacterial invasion of eukaryotic cells

“…The same cleavage pattern is observed upon incubation of actin with protealysin, a protease of S. proteamaculans homologous to grimelysin (Demidyuk et al, 2006;Tsaplina et al, 2009Tsaplina et al, , 2012.…”

“…The Gram-negative bacteria S. grimesii are facultative pathogens that cause nosocomial infections (Grimont & Grimont, 2006). we have detected grimelysin-homologous protease protealysin in the infected cells by staining the enzyme with specific antibodies (Tsaplina et al, 2012). However, at that time we could not exclude the possibility that protealysin was released from the damaged intracellular bacteria.…”

Bacterial Actin-Specific Endoproteases Grimelysin and Protealysin as Virulence Factors Contributing to the Invasive Activities of Serratia

“…The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by o-phenanthroline [19], i.e., has the same properties as grimelysin [8,18]. Protealysin and grimelysin (ECP 32) are also similar in their unique property of being able to digest actin specifically [8][9][10]22,33].…”

Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

“…Proteases found in Serratia proteamaculans and Serratia grimesii also have cytotoxic properties. S. pro-teamaculans strain 94 produces a 32-kDa thermostable protealysin that is able of cleaving filamentous actin and matrix metalloprotease MMP2 in human larynx carcinoma HEp-2 cells (28)(29)(30). Additionally, S. proteamaculans strain 94 is able to infect HEp-2 cells and was retained within approximately 10% of cells.…”