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.     

Bacterial Protease ECP32 Specifically Hydrolyzing Actin and Its Effect on Cytoskeleton in vivo

A procedure for isolation of bacterial protease ECP32 yielding 100 µg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.     

Express-method of obtaining protease ECP32, a unique instrument in actin investigations

In studies of actin, the basic protein of muscles and cytoskeleton, protease ECP32 is of particular significance. This enzyme originates from the natural enterobacteria strain that accumulates minor amounts of the protease intracellularly at the post-exponential growth phase. The limiting factor for this biosynthesis is the amount of oxygen that enters the medium. The highly efficient method of the two-phase cultivation with vigorous aeration at the exponential growth phase was recommended. Due to the studied enzyme properties, the use of either the affinity or one-stage purification methods results in a rather decreased potential for success. To overcome the obstacles of the above methods, we developed a simple method for the ECP32 preparation and storage with purity and activity levels that satisfy the requirements of the actin structure and function investigations.     

Selective blockage of Serratia marcescens ShlA by nickel inhibits the pore‐forming toxin‐mediated phenotypes in eukaryotic cells

Serratia marcescens is an opportunistic pathogen with increasing incidence in clinical settings. This is mainly attributed to the timely expression of a wide diversity of virulence factors and intrinsic and acquired resistance to antibiotics, including β‐lactams, aminoglycosides, quinolones, and polypeptides. For these reasons, S. marcescens has been recently categorised by the World Health Organization as one priority to strengthen efforts directed to develop new antibacterial agents. Therefore, it becomes critical to understand the underlying mechanisms that allow Serratia to succeed within the host. S. marcescens ShlA pore‐forming toxin mediates phenotypes that alter homeostatic and signal transduction pathways of host cells. It has been previously demonstrated that ShlA provokes cytotoxicity, haemolysis and autophagy and also directs Serratia egress and dissemination from invaded nonphagocytic cells. However, molecular details of ShlA mechanism of action are still not fully elucidated. In this work, we demonstrate that Ni²⁺ selectively and reversibly blocks ShlA action, turning wild‐type S. marcescens into a shlA mutant strain phenocopy. Combined use of Ni²⁺ and calcium chelators allow to discern ShlA‐triggered phenotypes that require intracellular calcium mobilisation and reveal ShlA function as a calcium channel, providing new insights into ShlA mode of action on target cells.     

Septin‐regulated actin dynamics promote Salmonella invasion of host cells

Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells.     

Girdin,an actin‐binding protein,is critical for migration,adhesion, and invasion of human glioblastoma cells

Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration.

     

RECK‐mediated inhibition of glioma migration and invasion

RECK is an anti‐tumoral gene whose activity has been associated with its inhibitory effects regulating MMP‐2, MMP‐9, and MT1‐MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP‐2, MMP‐9, and MT1‐MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin–myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P‐FAK) in mature focal adhesions associated with stress fibers; whereas P‐FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor‐suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52–61, 2010. © 2010 Wiley‐Liss, Inc.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Apple S‐RNase interacts with an actin‐binding protein,MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

In S‐RNase‐mediated self‐incompatibility, S‐RNase secreted from the style destroys the actin cytoskeleton of the self‐pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S‐RNase does not bind or sever filamentous actin (F‐actin). In apple (Malus domestica), we identified an actin‐binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S‐RNase inhibits the F‐actin‐severing activity of MdMVG in vitro. In vivo, the addition of S‐RNase to self‐pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self‐pollination induction in the presence of MdMVG overexpression. By generating 25 single‐, double‐ and triple‐point mutations in the amino acid motif E‐E‐K‐E‐K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple‐point mutant, MdMVG^{(E167A/E171A/K185A)}, that no longer has F‐actin‐severing activity or interacts with any of the four S‐haplotype S‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S‐RNases. We conclude that apple S‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.     

Inhibition of GGTase‐I and FTase disrupts cytoskeletal organization of human PC‐3 prostate cancer cells

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC‐3 prostate cancer cells. This was done by using FTI‐277, GGTI‐298 or NE‐10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)‐I and ‐II, respectively. Treatment of PC‐3 cells with GGTI‐298 and FTI‐277 inhibited migration and invasion in a time‐ and dose‐dependent manner. This was associated with disruption of F‐actin organization and decreased recovery of GFP–actin. Immunoblot analysis of various cytoskeleton‐associated proteins showed that the most striking change in GGTI‐298‐ and FTI‐277‐treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC‐3 cells with GGTI‐298 also affected the dynamics of GFP–paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p‐21‐associated kinase)‐2 were also lowered by GGTI‐298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI‐298 had a minor effect on the activity of MMP‐9. RNAi knockdown of GGTase‐Iβ inhibited invasion, disrupted F‐actin organization and decreased the level of cofilin in PC‐3 cells. NE‐10790 did not have any effect on PC‐3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase‐I‐ and FTase‐catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.     

Sialic acid‐dependent interaction of group B streptococci with influenza virus‐infected cells reveals a novel adherence and invasion mechanism

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3‐linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3‐linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co‐sedimentation assay revealed that viruses with a preference for α2,3‐linked sialic acids bind to GBS in a sialic acid‐dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination‐inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre‐infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co‐infected by influenza viruses. This co‐infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3‐linked sialic acids and human viruses binding to this linkage type.     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.