RESUMEN
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.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Serratia/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Acetilcisteína/farmacología , Línea Celular Tumoral , HumanosRESUMEN
Facultative pathogens Serratia grimesii are able to invade eukaryotic cells where they have been found in vacuoles and free in the cytoplasm (Efremova et al., 2001; Bozhokina et al., 2011). However, efficiency of this invasion is low, and the mechanisms of the invasion related to the initial steps of the process are not known. In the present study, we have increased the invasion efficiency by incubation of HeLa cells with N-acetylcysteine (NAC) preceding the infection. In the NAC-pretreated cells, two modes of S. grimesii to enter HeLa cells were observed. In the most cases, the penetration of S. grimesii into the cell was consistent with the "zipper mechanism", involving specific interaction of bacterial invasin with a host cell surface receptor. However, in some cases, bacteria were trapped by membrane ruffling probably produced by injected bacterial proteins that trigger the bacterial uptake process, as described in the "trigger mechanism". Further elucidation of bacterial and cellular factors involved in the bacteria-host cell interaction should clarify whether two different mechanisms or a predominant one operate during S. grimesii invasion.
Asunto(s)
Citoplasma/ultraestructura , Células Eucariotas/ultraestructura , Interacciones Huésped-Patógeno , Serratia/ultraestructura , Acetilcisteína/farmacología , Adhesinas Bacterianas/metabolismo , Citoplasma/efectos de los fármacos , Células Eucariotas/efectos de los fármacos , Células HeLa , Humanos , Microscopía Electrónica , Serratia/metabolismo , Serratia/patogenicidadRESUMEN
In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called "comet-like tail", is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.
Asunto(s)
Actinas/metabolismo , Transporte Biológico , Polimerizacion , Actinas/química , Animales , Bacterias/metabolismo , Vesículas Citoplasmáticas/metabolismo , Dinaminas/metabolismo , Humanos , Microtúbulos/metabolismo , MovimientoRESUMEN
It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ~1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.
Asunto(s)
Actinas/química , Endopeptidasas/química , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/enzimología , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Bacillus subtilis/enzimología , Candida albicans/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/inmunología , Bacterias Gramnegativas/enzimología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Sueros Inmunes , Saccharomyces cerevisiae/enzimología , Análisis de Secuencia de ProteínaRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Células Eucariotas/microbiología , Metaloproteasas/metabolismo , Metaloproteínas/metabolismo , Serratia/enzimología , Actinas/aislamiento & purificación , Animales , Adhesión Bacteriana , Proteínas Bacterianas/aislamiento & purificación , Línea Celular , Técnicas de Cocultivo , Endocitosis , Escherichia coli/enzimología , Células Eucariotas/ultraestructura , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteasas/aislamiento & purificación , Metaloproteínas/aislamiento & purificación , Metaloproteínas/fisiología , Músculo Esquelético/química , Conejos , Serratia/patogenicidad , Serratia/ultraestructura , Especificidad por Sustrato , Termolisina/metabolismoRESUMEN
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.
Asunto(s)
Acetilcisteína/farmacología , Actinas/metabolismo , Transformación Celular Viral/efectos de los fármacos , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Glutatión/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células 3T3 BALB , Línea Celular Transformada , Transformación Celular Viral/fisiología , Escherichia coli/efectos de los fármacos , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Virus 40 de los SimiosRESUMEN
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.