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

The article reviews the discovery, properties and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of and protealysin of , characterized by both a highly specific "actinase" activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization.

Functional impact of cholesterol sequestration on actin cytoskeleton in normal and transformed fibroblasts.

Membrane cholesterol and lipid rafts are implicated in various signalling processes involving actin rearrangement in living cells. However, functional link between raft integrity and organisation of cytoskeleton remains unclear. We have compared the effect of cholesterol sequestration on F-actin structures in normal and transformed fibroblasts in which microfilament system is developed to a different extent.

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

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 Int35, 111-118]. The proteases have been characterized as actin-hydrolyzing enzymes with a narrow specificity toward intact cell proteins.

Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin.

Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm.

N-acetylcysteine-induced changes in susceptibility of transformed eukaryotic cells to bacterial invasion.

The effect of N-acetylcysteine (NAC) on morphological and physiological properties of "normal" 3T3 and 3T3-SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3-SV40 fibroblasts demonstrated formation of well-adhered cells with structured 3T3-like stress-fibers.