RESUMEN
We have previously shown that during late stages of the infectious process, serogroup B meningococci (MenB) are able to escape the phagosome of in vitro-infected human epithelial cells. They then multiply in the cytosolic environment and spread intracellularly and to surrounding cells by exploiting the microtubule cytoskeleton, as suggested by results of infections in the presence of microtubule inhibitors and evidence of nanotubes connecting neighboring cells. In this study, by using microtubule binding assays with purified microtubule asters and bundles and microtubule bundles synthesized in vitro, we demonstrate that the MenB capsule directly mediates the interaction between bacteria and microtubules. The direct interaction between the microtubules and the MenB capsular polysaccharide was confirmed by coimmunoprecipitation experiments. Unexpectedly, serogroup C meningococci (MenC), which have a capsular polysaccharide that differs from that of MenB only by its anomeric linkage, α(2â9) instead of α(2â8), were not able to interact with the microtubules, and the lack of interaction was not due to capsular polysaccharide O-acetylation that takes place in most MenC strains but not in MenB strains. Moreover, we demonstrate that the MenB capsular polysaccharide inhibits tubulin polymerization in vitro. Thus, at variance with MenC, MenB may interfere with microtubule dynamics during cell infection.
Asunto(s)
Cápsulas Bacterianas/inmunología , Interacciones Huésped-Patógeno/inmunología , Infecciones Meningocócicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Tubulina (Proteína)/inmunología , Adhesión Bacteriana/fisiología , Cápsulas Bacterianas/fisiología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Microtúbulos/inmunología , PolimerizacionRESUMEN
Charcot-Marie-Tooth (CMT) type 2 neuropathies are a group of autosomal-dominant axonal disorders genetically and clinically heterogeneous. In particular, CMT type 2B (CMT2B) neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Recently, four missense mutations in the small GTPase Rab7 associated with the Charcot-Marie Tooth type 2B phenotype have been identified. These mutations target highly conserved amino acid residues. However, nothing is known about whether and how these mutations affect Rab7 function. We investigated the biochemical and functional properties of three of the mutant proteins. Interestingly, all three proteins exhibited higher nucleotide exchange rates and hydrolyzed GTP slower than the wild-type protein. In addition, whereas 23% of overexpressed wild-type Rab7 was GTP bound in HeLa cells, the large majority of the mutant proteins (82-89%) were in the GTP-bound form, consistent with the data on GTP hydrolysis and exchange rates. The CMT2B-associated Rab7 proteins were also able to bind the Rab7 effector RILP (Rab-interacting lysosomal protein) and to rescue Rab7 function after silencing. Altogether, these data demonstrate that all tested CMT2B-associated Rab7 mutations are mechanistically similar, suggesting that activated forms of the Rab7 are responsible for CMT2B disease.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Mutación Missense , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Endocitosis , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas Mutantes/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión a GTP rab7RESUMEN
In this study we used HeLa cells to investigate the role of the HrpB-HrpA two-partner secretion (TPS) system in the meningococcal infection cycle. Although there is evidence that several pathogenic microorganisms may use TPS systems to colonize epithelial surfaces, the meningococcal HrpB-HrpA TPS system was not primarily involved in adhesion to or invasion of HeLa cells. Instead, this system was essential for intracellular survival and escape from infected cells. Gentamicin protection assays, immunofluorescence and transmission electron microscopy analyses demonstrated that, in contrast to the wild-type strain, HrpB-HrpA-deficient mutants were primarily confined to late endocytic vacuoles and trapped in HeLa cells. Haemolytic tests using human erythrocytes suggested that the secreted HrpA proteins could act as manganese-dependent lysins directly involved in mediating vacuole escape. In addition, we demonstrated that escape of wild-type meningococci from infected cells required the use of an intact tubulin cytoskeleton and that the hrpB-hrpA genes, which are absent in other Neisseria spp., were upregulated during infection.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Viabilidad Microbiana , Neisseria meningitidis/fisiología , Factores de Virulencia/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Recuento de Colonia Microbiana/métodos , Citoesqueleto/fisiología , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Gentamicinas/farmacología , Células HeLa , Hemólisis , Humanos , Microscopía Electrónica de Transmisión , Neisseria meningitidis/genética , Vacuolas/microbiología , Factores de Virulencia/genéticaRESUMEN
Four missense mutations, that target highly conserved amino acid residues in the small GTPase Rab7, have been associated with the Charcot-Marie-Tooth (CMT) type 2B phenotype. CMT2B peripheral axonal neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Here, we have investigated the biochemical and functional properties of the Rab7 K157N mutated protein. Interestingly, Rab7 K157N showed altered nucleotide exchange rate and GTP hydrolysis compared to the wild type protein. Consistently, the majority of the expressed protein in HeLa cells was bound to GTP. In addition, Rab7 K157N was able to restore EGF degradation, previously inhibited by Rab7 silencing. Altogether these data indicate that Rab7 K157N, similarly to the other three mutated proteins causative of CMT2B, is predominantly in the GTP-bound form and behaves as an active mutant. Therefore, activated forms of Rab7 protein cause the CMT2B disease.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/enzimología , Mutación Missense , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Asparagina/química , Asparagina/genética , Proteínas de Ciclo Celular/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Secuencia Conservada , Receptores ErbB/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Lisina/química , Lisina/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , Proteínas de Unión al GTP rab/química , Proteínas de Unión a GTP rab7RESUMEN
Rab proteins are master regulators of vesicular membrane traffic of endocytic and exocytic pathways. They basically serve to recruit proteins and lipids required for vesicle formation, docking, and fusion. Each Rab protein is able to recruit one or more effectors, and, through the action of effectors, it drives its specific downstream functions. The Rab interacting lysosomal protein (RILP) is a common effector of Rab7 and Rab34, two Rab proteins implicated in the biogenesis of lysosomes. RILP is recruited onto late endosomal/lysosomal membranes by Rab7-GTP where it induces the recruitment of the dynein-dynactin motor complexes. Therefore, through the timed and selective dynein motor recruitment onto late endosomes and lysosomes, Rab7 and RILP control transport to endocytic degradative compartments. A similar role for Rab7 and RILP has been demonstrated also for phagosomes. Indeed, RILP recruits dynein-dynactin motors on Rab7-GTP-positive phagosomes and the recruitment not only displaces phagosomes centripetally, but also promotes the extension of phagosomal tubules toward late endocytic compartments. RILP is therefore a key protein for the biogenesis of lysosomes and phagolysosomes. This chapter describes how to express wild-type or mutated RILP in mammalian cells, and how to test the effects caused by RILP dysfunction. In particular, we report assays to monitor the interaction between RILP and Rab7, morphology and distribution of endosomes, and to measure degradation of endocytic markers.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Proteínas Portadoras/genética , Cartilla de ADN , Vectores Genéticos , Células HeLa , Humanos , Lipoproteínas LDL/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Orgánulos/metabolismoRESUMEN
While much data exist in the literature about how Neisseria meningitidis adheres to and invades human cells, its behavior inside the host cell is largely unknown. One of the essential meningococcal attributes for pathogenesis is the polysaccharide capsule, which has been shown to be important for bacterial survival in extracellular fluids. To investigate the role of the meningococcal capsule in intracellular survival, we used B1940, a serogroup B strain, and its isogenic derivatives, which lack either the capsule or both the capsule and the lipooligosaccharide outer core, to infect human phagocytic and nonphagocytic cells and monitor invasion and intracellular growth. Our data indicate that the capsule, which negatively affects bacterial adhesion and, consequently, entry, is, in contrast, fundamental for the intracellular survival of this microorganism. The results of in vitro assays suggest that an increased resistance to cationic antimicrobial peptides (CAMPs), important components of the host innate defense system against microbial infections, is a possible mechanism by which the capsule protects the meningococci in the intracellular environment. Indeed, unencapsulated bacteria were more susceptible than encapsulated bacteria to defensins, cathelicidins, protegrins, and polymyxin B, which has long been used as a model compound to define the mechanism of action of CAMPs. We also demonstrate that both the capsular genes (siaD and lipA) and those encoding an efflux pump involved in resistance to CAMPs (mtrCDE) were up-regulated during the intracellular phase of the infectious cycle.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Macrófagos/microbiología , Neisseria meningitidis Serogrupo B/crecimiento & desarrollo , Neisseria meningitidis Serogrupo B/patogenicidad , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Adhesión Bacteriana , Cápsulas Bacterianas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Células HeLa/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Neisseria meningitidis Serogrupo B/efectos de los fármacos , Neisseria meningitidis Serogrupo B/genética , Células U937/microbiologíaRESUMEN
The Rab-interacting lysosomal protein (RILP) has been identified as an effector for the small GTPases Rab7 and Rab34. It has been demonstrated that Rab7 and RILP are key proteins for the biogenesis of lysosomes and phagolysosomes. Indeed, expression of dominant negative mutants of Rab7 or of the C-terminal half of RILP impairs biogenesis and function of these organelles. In this study we have isolated, using the yeast two-hybrid system, the EAP30/SNF8/VPS22 subunit of the ESCRT-II complex as a RILP interacting protein. We demonstrated that VPS22 interacts with the N-terminal half of RILP. The interaction data obtained with the two-hybrid system were confirmed by co-immunoprecipitation. In addition, confocal immunofluorescence revealed colocalization of GFP-RILP and HA-VPS22. These data suggest that RILP could have a role in the biogenesis of multivesicular bodies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Fluorescente , Complejos Multiproteicos/metabolismo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Técnicas del Sistema de Dos HíbridosRESUMEN
GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in l-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the l-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.