RESUMEN
Historically, many species of bacteria have been reported to produce viable, cell wall deficient (CWD) variants. A variety of terms have been used to refer to CWD bacteria and a plethora of methods described in which to induce, cultivate and propagate them. In this review, we will examine the long history of scientific research on CWD bacteria examining the methods by which CWD bacteria are generated; the requirements for survival in a CWD state; the replicative processes within a CWD state; and the reversion of CWD bacteria into a walled state, or lack thereof. In doing so, we will present evidence that not all CWD variants are alike and that, at least in some cases, CWD variants arise through an adaptive lifestyle switch that enables them to live and thrive without a cell wall, often to avoid antimicrobial activity. Finally, the implications of CWD bacteria in recurring infections, tolerance to antibiotic therapy and antimicrobial resistance will be examined to illustrate the importance of greater understanding of the CWD bacteria in human health and disease.
Asunto(s)
Bacterias , Pared Celular , Antibacterianos/farmacología , Bacterias/genética , Humanos , Estilo de VidaRESUMEN
Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.
Asunto(s)
Bacteriófagos/fisiología , Membrana Celular/virología , Escherichia coli/virología , Vesículas Extracelulares/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vesículas Extracelulares/genéticaRESUMEN
Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , ADN Bacteriano/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Bacteriólisis , Endopeptidasas/genética , Endopeptidasas/metabolismo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Mutación , Pseudomonas aeruginosa/metabolismoRESUMEN
Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Forma de la Célula , Haloferax volcanii/citología , Haloferax volcanii/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , División Celular , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Movimiento , Tubulina (Proteína)/químicaRESUMEN
Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3'-5'-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa.
Asunto(s)
Biopelículas/crecimiento & desarrollo , AMP Cíclico/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/metabolismo , ADN Bacteriano , Interacciones Huésped-Patógeno , Movimiento/efectos de los fármacos , Mucinas/farmacología , Oligopéptidos/farmacología , Infecciones por Pseudomonas/microbiología , Eliminación de Secuencia , Albúmina Sérica/farmacología , Transducción de Señal , Factores de Virulencia/metabolismoRESUMEN
Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa.
Asunto(s)
Biopelículas , Pseudomonas aeruginosa/fisiología , Transformación Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , ADN/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/genéticaRESUMEN
Object tracking is an instrumental tool supporting studies of cellular trafficking. There are three challenges in object tracking: the identification of targets; the precise determination of their position and boundaries; and the assembly of correct trajectories. This last challenge is particularly relevant when dealing with densely populated images with low signal-to-noise ratios-conditions that are often encountered in applications such as organelle tracking, virus particle tracking or single-molecule imaging. We have developed a set of methods that can handle a wide variety of signal complexities. They are compiled into a free software package called Diatrack. Here we review its main features and utility in a range of applications, providing a survey of the dynamic imaging field together with recommendations for effective use. The performance of our framework is shown to compare favorably to a wide selection of custom-developed algorithms, whether in terms of localization precision, processing speed or correctness of tracks.
Asunto(s)
Algoritmos , Transporte de Proteínas/fisiología , Imagen Individual de Molécula , Programas Informáticos , Humanos , Microscopía Fluorescente/métodosRESUMEN
Pandoraea species have been isolated from diverse environmental samples and are emerging important respiratory pathogens, particularly in people with cystic fibrosis (CF). In the present study, two bacterial isolates initially recovered from consecutive sputum samples collected from a CF patient and identified as Pandoraea pnomenusa underwent a polyphasic taxonomic analysis. The isolates were found to be Gram-negative, facultative anaerobic motile bacilli and subsequently designated as strains 6399T (=LMG29626T=DSM103228T) and 7641 (=LMG29627=DSM103229), respectively. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences revealed that 6399T and 7641 formed a distinct phylogenetic lineage within the genus Pandoraea. Genome sequence comparison analysis indicated that strains 6399T and 7641 are clonal and share 100â% similarity, however, similarity to other type strains (ANIb 73.2-88.8â%, ANIm 83.5-89.9â% and OrthoANI 83.2-89.3â%) indicates that 6399T and 7641 do not belong to any of the reported type species. The major cellular fatty acids of 6399T were C16â:â0 (32.1â%) C17â:â0cyclo (18.7â%) and C18â:â1ω7c (14.5â%), while Q-8 was the only respiratory quinone detected. The major polar lipids identified were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of 6399T was 62.9 (mol%). Strain 6399T can be differentiated from other members of Pandoraea by the absence of C19â:â0ω8c cyclo and by the presence of C17â:â0ω8c cyclo. Together our data show that the bacterial strains 6399T and 7641 represent a novel species of the genus Pandoraea, for which the name Pandoraea fibrosis sp. nov. is proposed (type strain 6399T).
Asunto(s)
Burkholderiaceae/clasificación , Filogenia , Esputo/microbiología , Técnicas de Tipificación Bacteriana , Composición de Base , Burkholderiaceae/aislamiento & purificación , Fibrosis Quística , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Humanos , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Tasmania , Ubiquinona/químicaRESUMEN
Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Red trans-Golgi/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células COS , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Especificidad de Órganos , Páncreas/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.
Asunto(s)
Carcinogénesis/metabolismo , Proteínas del Helminto/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Opistorquiasis/complicaciones , Opisthorchis/metabolismo , Cicatrización de Heridas/fisiología , Secuencia de Aminoácidos , Animales , Neoplasias de los Conductos Biliares/parasitología , Colangiocarcinoma/parasitología , Humanos , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Opistorquiasis/metabolismo , Progranulinas , Interferencia de ARNRESUMEN
Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.
Asunto(s)
Antibacterianos/aislamiento & purificación , Pseudomonas aeruginosa/química , Piocinas/aislamiento & purificación , Animales , Antibacterianos/química , Antibacterianos/farmacología , Dicroismo Circular , Clonación Molecular , Enfermedades Pulmonares/tratamiento farmacológico , Ratones , Piocinas/química , Piocinas/farmacología , Dispersión del Ángulo Pequeño , Espectrofotometría Ultravioleta , Difracción de Rayos XRESUMEN
Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Mycoplasma hyopneumoniae/metabolismo , Procesamiento Proteico-Postraduccional , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Cromatografía de Afinidad , Electroforesis en Gel Bidimensional , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Immunoblotting , Inmunohistoquímica , Datos de Secuencia Molecular , Mycoplasma hyopneumoniae/genética , Polisacáridos/metabolismo , Análisis por Matrices de Proteínas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Espectrometría de Masas en TándemRESUMEN
Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.
Asunto(s)
Biopelículas , ADN Bacteriano/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMEN
BACKGROUND: Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. METHODS: Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization. RESULTS: EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes. CONCLUSIONS: This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Endocitosis , Células Epiteliales/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Opisthorchis/metabolismo , Animales , Bilis/química , Cricetinae , Células Epiteliales/fisiología , Vesículas Extracelulares/química , Humanos , Espectrometría de Masas , Microscopía , Opistorquiasis/parasitología , Opistorquiasis/patología , Fenotipo , Proteoma/análisisRESUMEN
In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a sub-nuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex (NPC). However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging, we show that the dynamic localization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochormatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and chromatin immunoprecipitation analyses revealed that, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used peptide nucleic acids (PNA) to downregulate PfSec13 and show that it is essential for parasite proliferation in human erythrocytes.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina/metabolismo , Eritrocitos/parasitología , Malaria Falciparum/parasitología , Microtúbulos/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Plasmodium falciparum/patogenicidad , Secuencia de Aminoácidos , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Malaria Falciparum/fisiopatología , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Oligonucleótidos Antisentido/genética , Plasmodium falciparum/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Transgenes/genéticaRESUMEN
Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Virus Vaccinia/fisiología , Vaccinia/transmisión , Proteínas del Envoltorio Viral/metabolismo , Proteínas Estructurales Virales/metabolismo , Liberación del Virus/fisiología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Membrana Celular/ultraestructura , Membrana Celular/virología , Chlorocebus aethiops , Ensayo Cometa , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Fibroblastos/virología , Interacciones Huésped-Patógeno , Ratones , Microscopía Electrónica de Transmisión , Células 3T3 NIH , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Virus Vaccinia/ultraestructura , Células Vero , Proteínas del Envoltorio Viral/ultraestructura , Proteínas Estructurales Virales/ultraestructuraRESUMEN
BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa. RESULTS: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system. CONCLUSIONS: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furthermore, eATP produced by P. aeruginosa biofilms and by damaged epithelial cells may play a role in P. aeruginosa pathogenesis by inducing inflammatory damage and fibrosis. Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.
Asunto(s)
Adenosina Trifosfato/farmacología , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Fimbrias Bacterianas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Fimbrias Bacterianas/fisiología , Expresión Génica , Movimiento/efectos de los fármacos , Movimiento/fisiología , Pseudomonas aeruginosa/fisiología , Transducción de SeñalRESUMEN
FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.
Asunto(s)
Bacillus subtilis/citología , Proteínas Bacterianas/metabolismo , Citocinesis , Proteínas del Citoesqueleto/metabolismo , Imagenología Tridimensional/métodos , Microscopía/métodos , Staphylococcus aureus/citología , Bacillus subtilis/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Viabilidad Microbiana , Modelos Biológicos , Movimiento , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Staphylococcus aureus/metabolismo , Imagen de Lapso de TiempoRESUMEN
Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calpaína/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimología , Distrofia Muscular de Cinturas/metabolismo , Sarcoglicanopatías/metabolismo , Anexina A1/metabolismo , Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/fisiología , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Disferlina , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Proteínas Musculares/genética , Músculo Esquelético/citología , Músculo Esquelético/lesiones , Distrofia Muscular de Cinturas/patología , Fosfolípidos/metabolismo , Cultivo Primario de Células , Sarcoglicanopatías/patología , Proteínas de Motivos TripartitosRESUMEN
The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydiaâ CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.