Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells.

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MßCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MßCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MßCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MßCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MßCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MßCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion.

Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA.

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

The Role of SprIR Quorum Sensing System in the Regulation of Serratia proteamaculans 94 Invasion.

The bacteria Serratia proteamaculans 94 have a LuxI/LuxR type QS system consisting of AHL synthase SprI and the regulatory receptor SprR. We have previously shown that inactivation of the AHL synthase sprI gene resulted in an increase in the invasive activity of S. proteamaculans correlated with an increased bacterial adhesion. In the present work, the effects of inactivation of the S. proteamaculans receptor SprR are studied. Our results show that inactivation of the receptor sprR gene leads to an increase in bacterial invasion without any increase in their adhesion. On the other hand, inactivation of the sprR gene increases the activity of the extracellular protease serralysin. Inactivation of the QS system does not affect the activity of the pore-forming toxin ShlA and prevents the ShlA activation under conditions of a limited concentration of iron ions typical of the human body. While the wild type strain shows increased invasion in the iron-depleted medium, deletion of its QS system leads to a decrease in host cell invasion, which is nevertheless similar to the level of the wild type S. proteamaculans grown in the iron-rich medium. Thus, inactivation of either of the two component of the S. proteamaculans LuxI/LuxR-type QS system leads to an increase in the invasive activity of these bacteria through different mechanisms and prevents invasion under the iron-limited conditions.

Invasion of Serratia proteamaculans is regulated by the sprI gene encoding AHL synthase.

Quorum Sensing (QS) system regulates gene expression in response to a change in the density of the bacterial population. Facultative pathogen Serratia proteamaculans 94 has a LuxI/LuxR type QS system consisting of regulatory protein SprR and AHL synthase SprI. Invasive activity of these bacteria appears at the stationary growth phase corresponding to a maximal density of the bacterial population in vitro. To evaluate the contribution of QS system of S. proteamaculans 94 to the regulation of invasive activity, in this work, S. proteamaculans SprI(-) mutant carrying the inactivated AHL synthase gene was used. Inactivation of the AHL synthase sprI gene resulted in a more than fourfold increase in the invasive activity of S. proteamaculans preceded by the increased adhesion of bacteria to the cell surface. This effect correlated with the increased expression of the outer membrane protein ompX gene and the decrease in the activity of intrabacterial protease protealysin, whose substrate is OmpX. The inverse correlation between activity of protealysin and bacterial invasion was also observed in the model experiments under the iron-limiting culture conditions. These results show that QS system regulates the S. proteamaculans invasion. This regulation can involve changes both in the protealysin activity and in the level of the ompX gene transcription.

Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament.

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu2+ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg2+ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl2 strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2-3-fold lower than that in the MgCl2-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.

Cleavage of the outer membrane protein OmpX by protealysin regulates Serratia proteamaculans invasion.

Protealysin is a thermolysin-like protease of Serratia proteamaculans capable of specifically cleaving actin, which correlates with the invasive activity of these bacteria. Here, we show that inactivation of the protealysin gene does not inhibit invasion but, in contrast, leads to a twofold increase in the S. proteamaculans invasive activity. By mass spectrometry, we identified the outer membrane protein OmpX as a substrate of protealysin. Recombinant E. coli carrying the OmpX gene truncated by 40 N-terminal residues or both the OmpX and protealysin genes, in contrast to the full-length OmpX, do not increase adhesion of these bacteria, indicating that the 40 N-terminal residues of OmpX are indispensable for S. proteamaculans invasion. Our results show that both protealysin and its substrates can stimulate Serratia invasion.

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

Serratia grimesii are facultative pathogenic bacteria that can penetrate a wide range of host cells and cause infection, especially in immunocompromised patients. Previously, we have found that bacterial metalloprotease grimelysin is a potential virulence determinant of S. grimesii invasion (E. S. Bozhokina et al., (2011). Cell Biology International, 35(2), 111-118). Protease is characterized as an actin-hydrolyzing enzyme with a narrow specificity toward other cell proteins. It is not known, however, whether grimelysin is transported into eukaryotic cells. Here, we show, for the first time, that S. grimesii can generate outer membrane vesicles (OMVs) displayed specific proteolytic activity against actin, characteristic of grimelysin. The presence of grimelysin was also confirmed by the Western blot analysis of S. grimesii OMVs lysate. Furthermore, confocal microscopy analysis revealed that the S. grimesii grimelysin-containing OMVs attached to the host cell membrane. Finally, pretreatment of HeLa cells with S. grimesii OMVs before the cells were infected with bacteria increased the bacterial penetration several times. These data strongly suggest that protease grimelysin promotes S. grimesii internalization by modifying bacterial and/or host molecule(s) when it is delivered as a component of OMVs.

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

The article reviews the discovery, properties and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of Serratia grimesii and protealysin of Serratia proteamaculans, 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. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria Serratia grimesii and Serratia proteamaculans, producing grimelysin and protealysin, invade eukaryotic cells, and the recombinant Escherichia coli expressing the grimelysin or protealysins gene become invasive. Participation of the cellular c-Src and RhoA/ROCK signaling pathways in the invasion of eukaryotic cells by S. grimesii was shown, and involvement of E-cadherin in the invasion has been suggested. Moreover, membrane vesicles produced by S. grimesii were found to contain grimelysin, penetrate into eukaryotic cells and increase the invasion of bacteria into eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.

Cooperative effects of tropomyosin on the dynamics of the actin filament.

Tropomyosin (Tpm) plays an important role in regulating the organisation and functions of the actin cytoskeleton. Here, we describe a new approach to analyse the effects of Tpm on actin dynamics. Using F-actin proteolytically modified within the DNase-binding loop (ECP-actin), we show that Tpm binding almost completely suppresses the increased subunit exchange intrinsic for this F-actin. The effect is both concentration-dependent and cooperative, with half-maximal inhibition observed at about a 1 : 50 Tpm : actin ratio. Tpm decreases not only the number concentration of ECP-actin filaments, but also the rate of the filament subunit exchange. Our data suggest that Tpm regulates the dynamics of actin filaments by an allosteric strengthening of intermonomer contacts in the actin filament, and that this mechanism may be involved in the modulation of cytoskeletal dynamics.

Sodium fluoride as a nucleating factor for Mg-actin polymerization.

Dynamic instability of actin filaments can be inhibited by Pi analogs beryllium fluoride and aluminium fluoride that mimic the intermediate ADP-Pi state and stabilize actin filaments. On the other hand, the phosphoryl transfer enzymes can be activated in the absence of aluminium by magnesium fluoride if magnesium ions and sodium fluoride (NaF) were present in the solution. Whether magnesium fluoride promotes functional activities of actin is not known. Here we show, for the first time, that sodium fluoride strongly accelerates polymerization of highly dynamic Mg-F-actin assembled from the monomers proteolytically cleaved between Gly42 and Val43 within the D-loop with actin-specific protease protealysin (Pln-actin), apparently due to stabilization of nuclei formed at the initial step of actin polymerization. Thereby, NaF did not inhibit the ATPase activity (subunit exchange) on Pln-F-actin, did not increase the amount of Pln-F-actin sedimented by ultracentrifugation, and did not stabilize the inter-strand contacts of Pln-F-actin. On the other hand, NaF diminished accessibility of the nucleotide binding cleft of Mg-G-actin to trypsin, pointing to an additional cleft closure, and additionally protected the D-loop from the protealysin cleavage in Mg-F-actin, thus indicating that the longitudinal contacts are stabilized. We also demonstrate that in cultured cells NaF can directly promote assembly of F-actin structures under conditions when the corresponding activity of the RhoA pathway is inhibited. These data suggest that the NaF-induced assembly of actin filaments is promoted by magnesium fluoride that can be formed by the NaF-originating fluoride and the actin tightly bound magnesium.

Contribution of α-smooth muscle actin and extracellular matrix to the in vitro reorganization of cardiomyocyte contractile system.

Cardiomyocytes in culture undergo reversible rearrangement of their contractile apparatus with the conversion of typical myofibrils into the structures of non-muscle type and the loss of contractility. Along with these transformations, the cardiomyocytes gain the capacity to synthesize extracellular matrix. Here we show that during cultivation of rat neonatal cardiomyocytes, the inherent α-cardiac actin isoform is transiently replaced by α-smooth-muscle actin, whose expression is accompanied by transformation of myofibrils into stress-fiber-like structures. The following down-regulation of α-smooth muscle actin parallels restoration of myofibrillar system and correlates with the accumulation of extracellular collagen and laminin, initially missing from the cardiomyocytes culture.

Tropomyosin as a Regulator of Actin Dynamics.

Tropomyosin is a major regulatory protein of contractile systems and cytoskeleton, an actin-binding protein that positions laterally along actin filaments and modulates actin-myosin interaction. About 40 tropomyosin isoforms have been found in a variety of cytoskeleton systems, not necessarily connected with actin-myosin interaction and contraction. Involvement of specific tropomyosin isoforms in the regulation of key cell processes was shown, and specific features of tropomyosin genes and protein structure have been investigated with molecular biology and genetics approaches. However, the mechanisms underlying the effects of tropomyosin on cytoskeleton dynamics are still unclear. As tropomyosin is primarily an F-actin-binding protein, it is important to understand how it interacts both with actin and actin-binding proteins functioning in muscles and cytoskeleton to regulate actin dynamics. This review focuses on biochemical data on the effects of tropomyosin on actin assembly and dynamics, as well as on the modulation of these effects by actin-binding proteins. The data indicate that tropomyosin can efficiently regulate actin dynamics via allosteric conformational changes within actin filaments.

Dihydrolipoic but not alpha-lipoic acid affects susceptibility of eukaryotic cells to bacterial invasion.

Sensitivity of eukaryotic cells to facultative pathogens can depend on physiological state of host cells. Previously we have shown that pretreatment of HeLa cells with N-acetylcysteine (NAC) makes the cells 2-3-fold more sensitive to invasion by the wild-type Serratia grimesii and recombinant Escherichia coli expressing gene of actin-specific metalloprotease grimelysin [1]. To evaluate the impact of chemically different antioxidants, in the present work we studied the effects of α-Lipoic acid (LA) and dihydrolipoic acid (DHLA) on efficiency of S. grimesii and recombinant E. coli expressing grimelysin gene to penetrate into HeLa and CaCo cells. Similarly to the effect of NAC, pretreatment of HeLa and CaCo cells with 0.6 or 1.25 mM DHLA increased the entry of grimelysin producing bacteria by a factor of 2.5 and 3 for the wild-type S. grimesii and recombinant E. coli, respectively. In contrast, pretreatment of the cells with 0.6 or 1.25 mM LA did not affect the bacteria uptake. The increased invasion of HeLa and CaCo cells correlated with the enhanced expression of E-cadherin and ß-catenin genes, whereas expression of these genes in the LA-treated cells was not changed. Comparison of these results suggests that it is sulfhydryl group of DHLA that promotes efficient modification of cell properties assisting bacterial uptake. We assume that the NAC- and DHLA-induced stimulation of the E-cadherin-catenin pathway contributes to the increased internalization of the grimelysin producing bacteria within transformed cells.

Virulence factors contributing to invasive activities of Serratia grimesii and Serratia proteamaculans.

Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111-118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S. grimesii.

Draft Genome Sequence of Serratia grimesii Strain A2.

We report the first draft genome assembly of Serratia grimesii strain A2, previously identified as Escherichia coli strain A2, which produces protease ECP32 with a high specificity toward actin. S. grimesii strain A2 has multidrug resistance associated with a number of efflux pump genes.

The interaction of gelsolin with tropomyosin modulates actin dynamics.

We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of gelsolin. Limited proteolysis indicated that the loop connecting the gelsolin domains G3 and G4 is involved in tropomyosin binding. Under nonpolymerizing conditions, binding of tropomyosin neither prevented the formation of a 2: 1actin-gelsolin complex, nor did it affect the nucleating activity of gelsolin in actin polymerization, likely as a result of competitive displacement of tropomyosin from gelsolin. To evaluate the effect of tropomyosin on the actin filament severing activity of gelsolin, we measured both filamentous actin (F-actin) viscosity and the relative number concentrations of filaments after fragmentation, either by gelsolin alone or by gelsolin-tropomyosin complexes. The interaction of gelsolin with tropomyosin caused a reduction in F-actin severing activity of up to 80% compared to gelsolin alone. Thus, being bound to gelsolin, tropomyosin prevented gelsolin from severing actin filaments. By contrast, the severing activity of gelsolin for F-actin/tropomyosin was similar to that for F-actin alone even at a tropomyosin : actin saturation ratio of 1: 7. Thus, when bound to actin filaments, tropomyosin did not significantly inhibit the severing of filaments by gelsolin. The interaction between gelsolin and tropomyosin was largely independent of the muscle actin and tropomyosin isoforms investigated. The results obtained in the present study suggest that tropomyosin is involved in the modulation of actin dynamics not via the protection of filaments against severing, but rather by binding gelsolin in solution to prevent it from severing and to promote the formation of new actin filaments.

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.

N-acetylcysteine increases susceptibility of HeLa cells to bacterial invasion.

Serratia grimesii are non-pathogenic bacteria capable, however, to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin (Bozhokina et al. [2011] Cell. Biol. Int. 35: 111-118). To elucidate how invasion of grimelysin containing bacteria depends on physiological state of host cells, we studied the effect of N-acetylcysteine (NAC) on susceptibility of HeLa cells to invasion by the wild-type S. grimesii and recombinant E. coli expressing grimelysin gene. Incubation of HeLa cells with 10 mM NAC resulted in changes of cell morphology and disassembly of actin cytoskeleton that were reversed when NAC was removed from the culture medium. Both in the presence of NAC and upon its removal, the entry of grimelysin producing bacteria increased by a factor of 1.5-2 and 3-3.5 for wild-type S. grimesii and recombinant E. coli, respectively. This effect does not correlate with cytoskeleton rearrangements but may be due to the NAC-induced up-regulation of cell surface receptors playing a role in cell adhesion and cell-cell junctions. A twofold difference in the efficiency of S. grimesii and recombinant E. coli to enter the NAC-treated cells suggests that the entry of the wild-type and recombinant bacteria occurs via different receptors which activity is differently affected by NAC.

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.

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.