The Opposite Effects of ROCK and Src Kinase Inhibitors on Susceptibility of Eukaryotic Cells to Invasion by Bacteria Serratia grimesii.

Bacterial internalization into eukaryotic cells is ensured by a sophisticated interplay of bacterial and host cell factors. Being a part of cell environment, opportunistic intracellular bacteria have developed various mechanisms providing their interaction with cell surface receptors (E-cadherin, integrins, epidermal growth factor receptor), activation of components of eukaryotic signaling pathways, and facilitation of bacterial uptake, survival, and intracellular replication. Our previous studies on the mechanisms underlying penetration of the opportunistic bacteria Serratia grimesii into cultured eukaryotic cells have shown that pretreatment of the cells with N-acetylcysteine (NAC) promotes S. grimesii invasion, and this effect correlates with the upregulation of E-cadherin expression. Since NAC has been shown to regulate expression of both Src kinase and ROCK, the aim of this work was to reveal the role of these kinases in S. grimesii invasion. We demonstrated that Y-27632, a specific inhibitor of ROCK, significantly promoted invasion of cultured eukaryotic cells by S. grimesii. On the other hand, invasion of the same cells by S. grimesii was inhibited with the Src kinase inhibitor Src-I1 and siRNA directed against RhoA. The effects of the inhibitors correlated with the corresponding changes in the E-cadherin gene expression, upregulation by the ROCK inhibition and downregulation by the Src kinase inhibition. These results prove the participation of ROCK and Src protein kinases in the invasion of eukaryotic cells by the opportunistic pathogen S. grimesii, as well as suggest that other signaling pathways might be involved in S. grimesii uptake, that are promoted by the ROCK inhibition with Y-27632.

[Phagocytosis of bacterial pathogens: modification of cellular processes by bacterial factors].

Bacterial are able to invade eukaryotic cells manipulating their own uptake by the host cells phagocytosis, Invasive bacteria can induce this process using extracellular toxins, cell surface ligands and virulence factors injected into the host cell. Two main mechanisms of invasion are recognized: the "zipper" mechanism in which bacteria bind host cell receptors to initiate cytoskeletal rearrangements and membrane extensions necessary for invasion, and the trigger mechanism in which bacteria regulate their own phagocytosis injecting regulatory proteins into the host cell cytoplasm. Most often, the targets in the host cell are signaling pathways not specific for phagocytosis and directly cytoskeleton. In addition, phospholipid composition of the target cell membrane plays an important role in the regulation of bacterial invasion. Thus, the efficiency of invasion is determined not only by bacterial virulence factors, but also by the life cycle, transformation, composition of the membrane, and other physiological characteristics of the host cell. This review describes the various mechanism of bacterial invasion into eukaryotic cells and the factors that may determine the sensitivity of eukaryotic cells to the invasion.

Probing for actinase activity of protealysin.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.