RESUMEN

BACKGROUND AND PURPOSE: This study explored the possible occurrence of newly generated nerve cells in the ischemic cortex of adult rats after middle cerebral artery occlusion and reperfusion. METHODS: Nine- to 10-week-old male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion by the monofilament method. Rats received repeated intraperitoneal injections of the cell proliferation-specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Brain sections were processed for immunohistochemistry with an avidin-biotin complex-alkaline phosphatase and/or -peroxidase method. Brain sections processed with double-immunofluorescent staining were further scanned by confocal microscopy. RESULTS: Interspersed among the predominantly newly formed glial cells, some cells were double labeled by BrdU and 1 of the neuron-specific markers, Map-2, beta-tubulin III, and Neu N, at 30 and 60 days after stroke onset. These cells were randomly distributed throughout cortical layers II through VI, occurring with highest density in the ischemic boundary zone. Three-dimensional confocal analyses of BrdU and the neuron-specific marker Neu N confirmed their colocalization within the same cortical cells. CONCLUSIONS: This study suggests that new neurons can be generated in the cerebral cortex of adult rats after transient focal cerebral ischemia. Cortical neurogenesis may be a potential pathway for brain repair after stroke.

Asunto(s)

Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/fisiopatología , Infarto de la Arteria Cerebral Media/patología , Neuronas/citología , Regeneración , Animales , Bromodesoxiuridina/farmacocinética , Recuento de Células , División Celular , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Wistar , Reperfusión

RESUMEN

Pathogenic Yersinia species inject virulence proteins, known as Yops, into the cytosol of eukaryotic cells. The injection of Yops is mediated via a type III secretion system. Previous studies have suggested that YopE is targeted for secretion by two signals. One is mediated by its cognate chaperone YerA, whereas the other consists of either the 5' end of yopE mRNA or the N-terminus of YopE. In order to characterize the YopE N-terminal/5' mRNA secretion signal, the first 11 codons of yopE were systematically mutagenized. Frameshift mutations, which completely alter the amino acid sequence of residues 2-11 but leave the mRNA sequence essentially intact, drastically reduce the secretion of YopE in a yerA mutant. In contrast, a mutation that alters the yopE mRNA sequence, while leaving the amino acid sequence of YopE unchanged, does not impair the secretion of YopE. Therefore, the N-terminus of YopE, and not the 5' end of yopE mRNA, serves as a targeting signal for type III secretion. In addition, the chaperone YerA can target YopE for type III secretion in the absence of a functional N-terminal signal. Mutational analysis of the YopE N-terminus revealed that a synthetic amphipathic sequence of eight residues is sufficient to serve as a targeting signal. YopE is also secreted rapidly upon a shift to secretion-permissive conditions. This 'rapid secretion' of YopE does not require de novo protein synthesis and is dependent upon YerA. Furthermore, this burst of YopE secretion can induce a cytotoxic response in infected HeLa cells.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Mensajero/química , Transducción de Señal , Yersinia pseudotuberculosis/patogenicidad , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células HeLa/microbiología , Humanos , Immunoblotting , Datos de Secuencia Molecular , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo

RESUMEN

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Virulencia , Yersinia/enzimología , Yersinia/patogenicidad , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Activación Enzimática , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Microscopía Confocal , Datos de Secuencia Molecular , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Fibras de Estrés/metabolismo , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos Híbridos , Yersinia/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo

RESUMEN

The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.

Asunto(s)

Actinas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Yersinia pseudotuberculosis/crecimiento & desarrollo , Yersinia pseudotuberculosis/patogenicidad , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina/ultraestructura , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Cinética , Proteínas Recombinantes/metabolismo , Virulencia , Yersinia pseudotuberculosis/genética

RESUMEN

We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.

Asunto(s)

ADP Ribosa Transferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Difosfato Ribosa/metabolismo , Toxinas Bacterianas , Factor de Crecimiento Epidérmico/farmacología , Pseudomonas aeruginosa/enzimología , Proteínas ras/metabolismo , Células 3T3 , ADP Ribosa Transferasas/química , Animales , Sitios de Unión , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Yersinia pseudotuberculosis/fisiología

RESUMEN

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

Asunto(s)

Proteínas Bacterianas/metabolismo , Enteritis/microbiología , Células Eucariotas/microbiología , Salmonella/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Bovinos , Línea Celular , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Salmonella/genética , Salmonella/patogenicidad , Alineación de Secuencia , Virulencia

RESUMEN

Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Pseudomonas aeruginosa/genética , Yersinia pseudotuberculosis/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Secuencia Conservada , Eritrocitos/metabolismo , Eritrocitos/microbiología , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Células HeLa , Hemólisis , Humanos , Proteínas Tirosina Fosfatasas/metabolismo , Pseudomonas aeruginosa/patogenicidad , Análisis de Secuencia de ADN , Ovinos , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/metabolismo

RESUMEN

Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

Asunto(s)

Proteínas Bacterianas/genética , Líquidos Corporales/metabolismo , Ileítis/microbiología , Proteínas de la Membrana , Salmonella/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/fisiología , Transporte Biológico , Bovinos , Clonación Molecular , Células Epiteliales/microbiología , Expresión Génica/genética , Expresión Génica/fisiología , Genes Bacterianos , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Masculino , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo , Translocación Genética

RESUMEN

Exoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria-cell contact-dependent mechanism involving the ysc-encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.

Asunto(s)

ADP Ribosa Transferasas , Actinas/metabolismo , Toxinas Bacterianas , Fagocitosis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Pseudomonas aeruginosa/enzimología , Citoesqueleto de Actina/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Transporte Biológico , Citosol/metabolismo , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/toxicidad , Pseudomonas aeruginosa/inmunología , Yersinia pseudotuberculosis/metabolismo

RESUMEN

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Membrana Celular/metabolismo , Perros , Células Eucariotas/metabolismo , Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Yersinia pseudotuberculosis/genética

RESUMEN

During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/toxicidad , Transporte Biológico Activo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Eliminación de Gen , Genes Bacterianos , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Microscopía Confocal , Fagocitosis , Proteínas Tirosina Fosfatasas/metabolismo , Ovinos , Virulencia/genética , Virulencia/fisiología , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad

RESUMEN

The entry of Salmonella into cultured epithelial cells is dependent on genes located in several adjacent chromosomal loci. One of these loci encodes the recently identified secretory proteins, denoted Sips (Salmonella invasion proteins). SipB, C,D proteins are essential for the ability of the pathogen to invade epithelial cells. To examine if additional invasion-associated proteins were secreted by Salmonella dublin, the genes encoding already characterized secretory proteins were inactivated to facilitate this analysis. The proteins produced and secreted by a double fIIM/polar sipB mutant of S. dublin were analysed; this revealed a set of novel secreted proteins. These proteins, which we denoted Sops (Salmonella outer proteins), formed large filamentous aggregates in the medium of bacterial culture growing at 37 degrees C. These aggregates contained five predominant proteins. Here we report the identification and characterization of one of these proteins, SopE, which is a novel invasion-associated secretory protein of S. dublin. A specific sopE mutant of S. dublin was found to be defective for invasion into epithelial cells. Upon interaction of Salmonella with HeLa cells, SopE was found to be translocated into the cytoplasm of the target cell by extracellular bacteria. The translocation of SopE was shown to be dependent on the Sip proteins because a polar sipB mutant did not translocate SopE across the HeLa cell membrane.

Asunto(s)

Proteínas Bacterianas/metabolismo , Salmonella/patogenicidad , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Citoplasma/metabolismo , Genes Bacterianos , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Salmonella/genética , Salmonella/metabolismo , Virulencia

RESUMEN

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high-copy-number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild-type strain with respect to YopB-dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB-dependent translocation mechanism from surface-located bacteria and subsequently targeted to the inner surface of the target-cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.

Asunto(s)

Proteínas Bacterianas , Proteínas Serina-Treonina Quinasas/metabolismo , Yersinia pseudotuberculosis/enzimología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Expresión Génica , Células HeLa , Humanos , Mutación , Proteínas Serina-Treonina Quinasas/toxicidad

RESUMEN

YopH is translocated by cell-surface-bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild-type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell-associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.

Asunto(s)

Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Polaridad Celular , Proteínas Tirosina Fosfatasas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia de Bases , Transporte Biológico , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Macrófagos , Ratones , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Mutación , Fagocitosis , Proteínas Tirosina Fosfatasas/inmunología

RESUMEN

Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE-containing S. typhimurium expressed a YopE-mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Chaperonas Moleculares/metabolismo , Salmonella typhimurium/metabolismo , Shigella flexneri/metabolismo , Virulencia/fisiología , Yersinia pseudotuberculosis/metabolismo , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Células HeLa , Humanos , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/patogenicidad , Shigella flexneri/patogenicidad , Yersinia pseudotuberculosis/patogenicidad

RESUMEN

The virulence plasmid common to pathogenic Yersinia species encodes a number of secreted proteins denoted Yops (Yersinia outer proteins). Here, we identify and characterize a novel plasmid-encoded virulence determinant of Yersinia pseudotuberculosis, YopK. The yopK gene was found to be conserved among the three pathogenic Yersinia species and to be homologous to the previously described yopQ and yopK genes of Y. enterocolitica and Y. pestis, respectively. Similar to the other Yops, YopK expression and secretion were shown to be regulated by temperature and by the extracellular Ca2+ concentration; thus, yopK is part of the yop regulon. In addition, YopK secretion was mediated by the specific Yop secretion system. In Y. pseudotuberculosis, YopK was shown neither to have a role in this bacterium's ability to resist phagocytosis by macrophages nor to cause cytotoxicity in HeLa cells. YopK was, however, shown to be required for the bacterium to cause a systemic infection in both intraperitoneally and orally infected mice. Characterization of the infection kinetics showed that, similarly to the wild-type strain, the yopK mutant strain colonized and persisted in the Peyer's patches of orally infected mice. A yopE mutant which is impaired in cytotoxicity and in antiphagocytosis was, however, found to be rapidly cleared from these lymphoid organs. Neither the yopK nor the yopE mutant strain could overcome the primary host defense and reach the spleen. This finding implies that YopK acts at a different level during the infections process than the antiphagocytic YopE cytotoxin does.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/fisiología , Plásmidos , Yersinia pseudotuberculosis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Genes Reguladores , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fagocitosis , Conejos , Virulencia , Yersiniosis/etiología , Yersinia pseudotuberculosis/genética

RESUMEN

The virulence plasmid-encoded YopE cytotoxin of Yersinia pseudotuberculosis is secreted across the bacterial membranes and subsequently translocated into the eukaryotic cell. Translocation of YopE into target cells was recently shown to be polarized and only occurred at the zone of contact between the pathogen and the eukaryotic cell. Immunogold electron microscopy on cryosectioned Y. pseudotuberculosis revealed that YopE is secreted and deposited on the bacterial cell surface when the bacteria are grown in Ca(2+)-depleted media at 37 degrees C. No YopE was detected in the cytoplasm or in the membranes. In yerA mutants which are downregulated for YopE at a post-transcriptional level, the cytotoxin could only be detected in the cytoplasm. The overall recovery of YopE from the yerA mutant strain was, however, considerably lower than from the wild-type strain. yerA had no major effect on the translation of YopE, but was found to stabilize YopE in the cytoplasm. YerA was shown to specifically interact with YopE in the cytoplasm in vivo and this binding also correlated with YopE secretion. Targeting of YopE to the secretion loci as well as translocation of YopE into HeLa cells occurred also in the absence of YerA. Based on our findings, we suggest that YerA by binding to YopE stabilizes and maintains the cytotoxin in a secretion-competent conformation.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Transactivadores/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Transporte Biológico Activo/genética , Citoplasma/metabolismo , ADN Bacteriano/genética , Genes Bacterianos , Células HeLa , Humanos , Microscopía Inmunoelectrónica , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Transactivadores/genética , Virulencia , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/fisiología , Citotoxinas/fisiología , Transactivadores/fisiología , Yersinia pseudotuberculosis/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Clonación Molecular , Citotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Células HeLa , Humanos , Cinética , Luciferasas/genética , Mutación , Temperatura , Transactivadores/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico Activo , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células HeLa , Humanos , Inmunohistoquímica , Mutación , Proteínas Tirosina Fosfatasas/genética , Virulencia , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/fisiología , Yersinia pseudotuberculosis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Genes Bacterianos , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Fagocitosis , Plásmidos , Virulencia/genética , Virulencia/fisiología , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/fisiología