T. N. Efremova

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. The E. coli‐carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep‐2 cells. Moreover, internalization of non‐invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

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. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria‐susceptible 3T3‐SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC‐induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.

Specific invasion of transformed cells by Escherichia coli A2 strain.

Bacteria of the spontaneously isolated non‐pathogenic strain Escherichia coli A2 producing actin‐specific protease ECP 32 (Usmanova and Khaitlina, 1989) were shown to be taken up by transformed cells, whereas finite and immortal cell lines were resistant to the infection.

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.

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. The depletion of membrane cholesterol by methyl‐beta‐cyclodextrin (MbCD) resulted in a disruption of lipid rafts in plasma membrane as it was revealed by fluorescent labelling of GM1 ganglioside. In normal fibroblasts with highly developed microfilament system, the cholesterol depletion resulted in actin disassembly and reduction of stress fibres. However, in transformed cells containing low amount of fibrillar actin, MbCD treatment induced intensive formation of stress fibres and increased cell spreading. The results show that the effect of cholesterol depletion and lipid raft disruption on microfilament system is critically determined by the initial state of cytoskeleton, specifically, by the balance of polymerised and monomeric actin in the cell. We assume that uncapping of the microfilaments is the key step of cholesterol‐regulated actin remodelling.

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. 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. abolishes this effect and promotes the nucleation of protealysin‐cleaved Mg‐globular‐actin 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.

Decreased Sensitivity of Transformed 3T3-SV40 Cells Treated with N-Acetylcysteine to Bacterial Invasion

Long-term treatment of transformed 3T3-SV40 mouse fibroblasts with antioxidant N-acetylcysteine decreased cell level of ROS and increased the concentration of reduced glutathione. Removal of N-acetylcysteine from the medium led to the appearance of well-expressed stress fibrils, virtually absent in control cells. In contrast to control cells, these cells were not invaded by apathogenic Escherichia coli A2 strain producing ECP32 protease specifically cleaving actin. Antioxidant N-acetylcysteine can cause partial reversion of transformed phenotype at the expense of a shift of cell redox balance in favor of reduced glutathione.

Assembly of actin filaments induced by sequestration of membrane cholesterol in transformed cells

Cholesterol is a major lipid component of the plasma membrane that plays an important role in various signaling processes in mammalian cells. Our study is focused on the role of membrane cholesterol in the organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by a poorly developed actin network and less prominent stress fibers: human embryonic kidney HEK293, human epidermoid larynx carcinoma HEp-2, and mouse fibroblasts 3T3-SV40. Using Factin labeling with rhodamine phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin and polyene macrolide antibiotic filipin. The cells treated with these agents displayed similar reorganization of actin cytoskeleton involving filament assembly. In HEp-2 carcinoma cells and 3T3-SV40 fibroblasts, cholesterol-sequestering reagents induced intense stress fiber formation and enhanced cell spreading; i.e., features of transformed phenotype reversion were observed. The cytoskeleton rearrangements are probably initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.

Invasive Characteristics of Apathogenic Shigella flexneri 5a2c Mutant Obtained under the Effect of Furazolidone

Apathogenic Shigella flexneri 5a2c mutant treated with furazolidone can infect eucaryotic cells. These bacteria contain no virulence genes responsible for Sh. Flexneri invasion, which seems to be the cause of their apathogenicity. The capacity of bacteria to penetrate into eucaryotic cells correlates with the appearance of ECP 32 protease specifically cleaving actin.

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.