RESUMEN
The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes," which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Biopelículas , Farmacorresistencia Fúngica , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Lectinas de Unión a Manosa , Proteínas de la Membrana/metabolismo , Microscopía , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Ciego/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Listeria monocytogenes/patogenicidad , Proteínas de la Membrana/metabolismo , Animales , Ciego/microbiología , Línea Celular , Colon/microbiología , Modelos Animales de Enfermedad , Heces/microbiología , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/microbiología , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/metabolismo , Listeriosis/metabolismo , Listeriosis/microbiología , Ratones , Factores de Virulencia/metabolismoRESUMEN
Filopodia are thin cell extensions sensing the environment. They play an essential role during cell migration, cell-cell or cell-matrix adhesion, by initiating contacts and conveying signals to the cell cortex. Pathogenic microorganisms can hijack filopodia to invade cells by inducing their retraction towards the cell body. Because their dynamics depend on a discrete number of actin filaments, filopodia provide a model of choice to study elementary events linked to adhesion and downstream signalling. However, the determinants controlling filopodial sensing are not well characterized. In this study, we used beads functionalized with different ligands that triggered filopodial retraction when in contact with filopodia of epithelial cells. With optical tweezers, we were able to measure forces stalling the retraction of a single filopodium. We found that the filopodial stall force depends on the coating of the bead. Stall forces reached 8 pN for beads coated with the ß1 integrin ligand Yersinia Invasin, whereas retraction was stopped with a higher force of 15 pN when beads were functionalized with carboxyl groups. In all cases, stall forces increased in relation to the density of ligands contacting filopodial tips and were independent of the optical trap stiffness. Unexpectedly, a discrete and small number of Shigella type three secretion systems induced stall forces of 10 pN. These results suggest that the number of receptor-ligand interactions at the filopodial tip determines the maximal retraction force exerted by filopodia but a discrete number of clustered receptors is sufficient to induce high retraction stall forces.
Asunto(s)
Células Epiteliales/ultraestructura , Seudópodos/ultraestructura , Shigella/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos , Fenómenos Biomecánicos , Adhesión Celular , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Integrina beta1/metabolismo , Ligandos , Microscopía Confocal , Microesferas , Pinzas Ópticas , Unión Proteica , Seudópodos/microbiología , Seudópodos/fisiología , Análisis de la Célula Individual , Imagen de Lapso de TiempoRESUMEN
The human malaria parasite Plasmodium falciparum modifies the erythrocyte it infects by exporting variant proteins to the host cell surface. The var gene family that codes for a large, variant adhesive surface protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion. A single member of this gene family is highly transcribed while the other 59 members remain silenced. Importantly, var gene transcription occurs at a spatially restricted, but yet undefined, perinuclear site that is distinct from repressed var gene clusters. To advance our understanding of monoallelic expression, we investigated whether nuclear pores associate with the var gene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore, P. falciparum Nup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Colocalization studies of var transcripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and the var gene expression site in ring stage. Similar results were obtained for another differentially transcribed perinuclear gene family, the ribosomal DNA units. Furthermore, we show that in the poised state, the var gene locus is not physically linked to nuclear pores. Our results indicate that P. falciparum does form compartments of high transcriptional activity at the nuclear periphery which are, unlike the case in yeast, devoid of nuclear pores.
Asunto(s)
ADN Ribosómico/genética , Poro Nuclear/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Células Cultivadas , ADN Ribosómico/metabolismo , Eritrocitos/parasitología , Expresión Génica , Regulación de la Expresión Génica , Genes Protozoarios , Humanos , Proteínas de Complejo Poro Nuclear/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Trofozoítos/diagnóstico por imagen , Trofozoítos/metabolismo , UltrasonografíaRESUMEN
The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram-negative pathogen Salmonella typhimurium and the Gram-positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.
Asunto(s)
Flagelos/fisiología , Glicosiltransferasas/metabolismo , Helicobacter pylori/enzimología , Hexosaminidasas/metabolismo , Listeria monocytogenes/enzimología , Peptidoglicano/metabolismo , Salmonella typhimurium/enzimología , Helicobacter pylori/fisiología , Listeria monocytogenes/fisiología , Sustancias Macromoleculares/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Transporte de Proteínas , Salmonella typhimurium/fisiologíaRESUMEN
The molecular basis of the regulation of specific shapes and their role for the bacterial fitness remain largely unknown. We focused in this study on the Gram-negative and spiral-shaped Helicobacter pylori. To colonize its unique niche, H. pylori needs to reach quickly the human gastric mucosa, by swimming to and through the mucus layer. For that reason, the specific shape of H. pylori is predicted to be necessary for optimal motility in vivo, and consequently for its colonization ability. Here, we describe the involvement of a PG-modifying enzyme, HdpA (HP0506), in the mouse colonization ability of this bacterium, by regulating its shape. Indeed, the inactivation of the hp0506 gene led to a stocky and branched phenotype, affecting H. pylori colonization capacity despite a normal motility phenotype in vitro. In contrast, the overexpression of the hp0506 gene induced the transformation of H. pylori from rod to dividing cocci shaped bacteria. Furthermore, we demonstrated by PG analysis and enzymology, that HdpA carried both d,d-carboxypeptidase and d,d-endopeptidase activities. Thus, HdpA is the first enzyme belonging to the M23-peptidase family able to perform the d,d-carboxypeptidation and regulate cell shape.
Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/citología , Helicobacter pylori/patogenicidad , Metaloproteasas/metabolismo , Peptidoglicano/metabolismo , Factores de Virulencia/metabolismo , Animales , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Pared Celular/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Técnicas de Inactivación de Genes , Helicobacter pylori/enzimología , Metaloproteasas/genética , RatonesRESUMEN
Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow-generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.
Asunto(s)
Bacteriemia/microbiología , Adhesión Bacteriana/fisiología , Membrana Celular/metabolismo , Neisseria meningitidis/genética , Estrés Mecánico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Colesterol/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Hemodinámica , Humanos , Lípidos de la Membrana/metabolismo , Microscopía Electrónica , Neisseria meningitidis/fisiologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) efficiently propagates through cell-to-cell contacts, which include virological synapses (VS), filopodia, and nanotubes. Here, we quantified and characterized further these diverse modes of contact in lymphocytes. We report that viral transmission mainly occurs across VS and through "polysynapses," a rosette-like structure formed between one infected cell and multiple adjacent recipients. Polysynapses are characterized by simultaneous HIV clustering and transfer at multiple membrane regions. HIV Gag proteins often adopt a ring-like supramolecular organization at sites of intercellular contacts and colocalize with CD63 tetraspanin and raft components GM1, Thy-1, and CD59. In donor cells engaged in polysynapses, there is no preferential accumulation of Gag proteins at contact sites facing the microtubule organizing center. The LFA-1 adhesion molecule, known to facilitate viral replication, enhances formation of polysynapses. Altogether, our results reveal an underestimated mode of viral transfer through polysynapses. In HIV-infected individuals, these structures, by promoting concomitant infection of multiple targets in the vicinity of infected cells, may facilitate exponential viral growth and escape from immune responses.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/fisiología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Linfocitos T CD4-Positivos/ultraestructura , Femenino , Humanos , Células Jurkat , Macaca , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Seudópodos/virología , Replicación Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD(cl4) cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.
Asunto(s)
Células Epiteliales/microbiología , Escherichia coli/patogenicidad , Flagelos/fisiología , Interacciones Huésped-Patógeno , Túbulos Renales Colectores , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cistitis/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Flagelina/metabolismo , Humanos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/microbiología , Ratones , Pielonefritis/microbiología , Orina/microbiologíaRESUMEN
Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.
Asunto(s)
Aspergillus fumigatus/fisiología , Adhesión Celular , Galactosa/análogos & derivados , Galactosa/metabolismo , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/ultraestructura , Línea Celular , Células Epiteliales/microbiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/biosíntesis , Eliminación de Gen , Humanos , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Microscopía Electrónica de Rastreo , Micelio/ultraestructura , Esporas Fúngicas/crecimiento & desarrollo , Uridina Difosfato/análogos & derivados , Uridina Difosfato/biosíntesisRESUMEN
The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.
Asunto(s)
Adhesión Bacteriana , Enterocitos/inmunología , Enterocitos/microbiología , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/patogenicidad , Flagelos/fisiología , Interleucina-8/metabolismo , Brasil , Línea Celular , Recuento de Colonia Microbiana , Citoplasma/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli Enteropatógena/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Flagelina , Eliminación de Gen , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
Asunto(s)
Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Mycobacterium smegmatis/enzimología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Galactanos/química , Galactanos/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/aislamiento & purificación , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Datos de Secuencia Molecular , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismoRESUMEN
Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.
Asunto(s)
Apoptosis , Complejo IV de Transporte de Electrones/metabolismo , Lyssavirus/patogenicidad , Mitocondrias/fisiología , Mitocondrias/virología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Caspasa 9/metabolismo , Línea Celular , Cricetinae , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Humanos , Inmunoprecipitación , Lyssavirus/genética , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-beta(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-kappaB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Helicobacter pylori/fisiología , Lipoproteínas/fisiología , Secuencias de Aminoácidos , Amoxicilina/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Línea Celular , Forma de la Célula/fisiología , Pared Celular/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Prueba de Complementación Genética , Helicobacter pylori/citología , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Humanos , Lipoproteínas/genética , Mutación , Peptidoglicano/genética , Peptidoglicano/metabolismoRESUMEN
The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.
Asunto(s)
Genes Esenciales , Ingeniería Genética , Vectores Genéticos , Helicobacter pylori/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/fisiología , Datos de Secuencia Molecular , Mutagénesis , Regiones Promotoras GenéticasRESUMEN
Anosmin-1, encoded by the KAL-1 gene, is the protein defective in the X-linked form of Kallmann syndrome. This human developmental disorder is characterized by defects in cell migration and axon target selection. Anosmin-1 is an extracellular matrix protein that plays a role, in vitro, in processes such as cell adhesion, neurite outgrowth, axon guidance, and axon branching. The zebrafish possesses two orthologues of the KAL-1 gene: kal1a and kal1b, which encode anosmin-1a and anosmin-1b, respectively. Previous in situ hybridization studies have shown that kal1a and kal1b mRNAs are expressed in undetermined cells of the inner ear but not in neuromast cells. Using specific antibodies against anosmin-1a and anosmin-1b, we report here that both proteins are expressed in sensory hair cells of the inner ear cristae ampullaris and the lateral line neuromasts. Accumulation of these proteins was observed mainly at the level of the hair bundle and also at the cell membrane. In neuromast hair cells, immunogold scanning electronmicroscopy demonstrated that anosmin-1a and anosmin-1b were present at the surface of the stereociliary bundle. In addition, anosmin-1a, but not anosmin-1b, was detected on the track of the ampullary nerve. This is the first report of anosmin-1 expression in sensory hair cells of the inner ear and lateral line, and along the ampullary nerve track.
Asunto(s)
Oído Interno/química , Proteínas de la Matriz Extracelular/análisis , Células Ciliadas Auditivas/química , Sistema de la Línea Lateral/química , Proteínas del Tejido Nervioso/análisis , Células Receptoras Sensoriales/química , Pez Cebra/metabolismo , Animales , Oído Interno/anatomía & histología , Oído Interno/metabolismo , Proteínas de la Matriz Extracelular/genética , Técnica del Anticuerpo Fluorescente , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Inmunohistoquímica , Sistema de la Línea Lateral/citología , Sistema de la Línea Lateral/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/genética , Células Receptoras Sensoriales/metabolismo , Pez Cebra/anatomía & histología , Pez Cebra/genéticaRESUMEN
Helicobacter pylori, a human-specific bacterial pathogen responsible for severe gastric diseases, constitutes a major public health issue. In the last decade, rates of H. pylori resistance to antibiotics were increasing drastically, requiring alternative therapeutic strategies to deal with eradication failures. Therefore, we evaluated the potential of bulgecin A, a glycosidic inhibitor of the lytic transglycosylase (LTG) Slt70 of Escherichia coli, as a new therapeutic approach against the H. pylori infection. In this study, we show that bulgecin A is able to specifically inactivate the H. pylori LTG Slt, but not its ortholog MltD. Moreover, bulgecin A synergized with amoxicillin, an inhibitor of penicillin binding proteins, inducing strong morphological alterations, cellular damages, and cell death. Similarly, the simultaneous inactivation of the peptidoglycan (PG) peptidase HdpA and Slt led to inhibition of H. pylori growth, highlighting the strong potential of targeting the PG biosynthetic pathway at different biochemical steps to enhance our therapeutic approaches against bacteria. Hence, we propose that bulgecin A constitutes an attractive compound for the development of new therapeutic strategies against H. pylori combined with other inhibitors of PG biosynthetic enzymes.
Asunto(s)
Acetilglucosamina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Glicósido Hidrolasas/antagonistas & inhibidores , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Prolina/análogos & derivados , Acetilglucosamina/farmacología , Amoxicilina/farmacología , Animales , Antibacterianos/farmacología , Pared Celular/química , Sinergismo Farmacológico , Endopeptidasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Femenino , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Células HEK293 , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Peptidoglicano/biosíntesis , Prolina/farmacología , TransfecciónRESUMEN
Shigella, the causative agent of bacillary dysentery in humans, invades epithelial cells, using a type III secretory system (T3SS) to inject bacterial effectors into host cells and remodel the actin cytoskeleton. ATP released through connexin hemichanels on the epithelial membrane stimulates Shigella invasion and dissemination in epithelial cells. Here, we show that prior to contact with the cell body, Shigella is captured by nanometer-thin micropodial extensions (NMEs) at a distance from the cell surface, in a process involving the T3SS tip complex proteins and stimulated by ATP- and connexin-mediated signaling. Upon bacterial contact, NMEs retract, bringing bacteria in contact with the cell body, where invasion occurs. ATP stimulates Erk1/2 activation, which controls actin retrograde flow in NMEs and their retraction. These findings reveal previously unappreciated facets of interaction of an invasive bacterium with host cells and a prominent role for Erk1/2 in the control of filopodial dynamics.
Asunto(s)
Adenosina Trifosfato/metabolismo , Disentería Bacilar/enzimología , Disentería Bacilar/microbiología , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Seudópodos/microbiología , Shigella/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Disentería Bacilar/metabolismo , Disentería Bacilar/fisiopatología , Activación Enzimática , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Seudópodos/enzimología , Seudópodos/metabolismo , Shigella/genética , Transducción de SeñalRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) is a lymphotropic retrovirus whose cell-to-cell transmission requires cell contacts. HTLV-1-infected T lymphocytes form 'virological synapses', but the mechanism of HTLV-1 transmission remains poorly understood. We show here that HTLV-1-infected T lymphocytes transiently store viral particles as carbohydrate-rich extracellular assemblies that are held together and attached to the cell surface by virally-induced extracellular matrix components, including collagen and agrin, and cellular linker proteins, such as tetherin and galectin-3. Extracellular viral assemblies rapidly adhere to other cells upon cell contact, allowing virus spread and infection of target cells. Their removal strongly reduces the ability of HTLV-1-producing cells to infect target cells. Our findings unveil a novel virus transmission mechanism based on the generation of extracellular viral particle assemblies whose structure, composition and function resemble those of bacterial biofilms. HTLV-1 biofilm-like structures represent a major route for virus transmission from cell to cell.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Matriz Extracelular/virología , Infecciones por HTLV-I/transmisión , Virus Linfotrópico T Tipo 1 Humano/fisiología , Biopelículas , Concanavalina A , Productos del Gen env/metabolismo , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Microscopía Electrónica de Transmisión , Ensamble de Virus/fisiología , Acoplamiento Viral , Internalización del VirusRESUMEN
Septins are filament-forming GTPases implicated in several cellular functions, including cytokinesis. We previously showed that SEPT2, SEPT9, and SEPT11 colocalize with several bacteria entering into mammalian non-phagocytic cells, and SEPT2 was identified as essential for this process. Here, we investigated the function of SEPT11, an interacting partner of SEPT9 whose function is still poorly understood. In uninfected HeLa cells, SEPT11 depletion by siRNA increased cell size but surprisingly did not affect actin filament formation or the colocalization of SEPT9 with actin filaments. SEPT11 depletion increased Listeria invasion, and incubating SEPT11-depleted cells with beads coated with the Listeria surface protein InlB also led to increased entry as compared with control cells. Strikingly, as shown by fluorescence resonance energy transfer, the InlB-mediated stimulation of Met signaling remained intact in SEPT11-depleted cells. Taken together, our results show that SEPT11 is not required for the bacterial entry process and rather restricts its efficacy. Because SEPT2 is essential for the InlB-mediated entry of Listeria, but SEPT11 is not, our findings distinguish the roles of different mammalian septins.