RESUMEN
Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction.
Asunto(s)
Fimbrias Bacterianas , Sulfolobus acidocaldarius , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/metabolismo , Sulfolobus acidocaldarius/fisiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genéticaRESUMEN
Actin and actin-like proteins form filamentous polymers that carry out important cellular functions in all domains of life. In this review, we sketch a map of the function and regulation of actin-like proteins across bacteria, archaea, and eukarya, marking some of the terra incognita that remain in this landscape. We focus particular attention on archaea because mapping the structure and function of cytoskeletal systems across this domain promises to help us understand the evolutionary relationship between the (mostly) mono-functional actin-like filaments found in bacteria and the multi-functional actin cytoskeletons that characterize eukaryotic cells.
Asunto(s)
Actinas , Archaea , Actinas/metabolismo , Archaea/genética , Archaea/metabolismo , Citoesqueleto/metabolismo , Bacterias/metabolismo , Evolución BiológicaRESUMEN
Cell invasion is a multi-step process, initiated by the acquisition of a migratory phenotype and the ability to move through complex 3D extracellular environments. We determine the composition of cell-matrix adhesion complexes of invasive breast cancer cells in 3D matrices and identify an interaction complex required for invasive migration. ßPix and myosin18A (Myo18A) drive polarized recruitment of non-muscle myosin 2A (NM2A) to adhesion complexes at the tips of protrusions. Actomyosin force engagement then displaces the Git1-ßPix complex from paxillin, establishing a feedback loop for adhesion maturation. We observe active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. The recruitment of NM2A to adhesions creates a non-muscle myosin isoform gradient, which extends from the protrusion to the nucleus. We postulate that this gradient facilitates coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.
Asunto(s)
Citoesqueleto de Actina , Actomiosina , Retroalimentación , Movimiento Celular/fisiología , Actomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Adhesión Celular/fisiología , Matriz Extracelular/metabolismoRESUMEN
Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract. Here, we employ live-cell imaging under native conditions (75°C and pH 2), together with automated single-cell tracking, high-temperature fluorescence imaging, and genetic manipulation to demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on retractable adhesion pili. Our results demonstrate that archaeal adhesion pili are capable of retraction in the absence of a PilT retraction ATPase and suggests that the ancestral type IV pilus machinery in the last universal common ancestor (LUCA) relied on such a bifunctional ATPase for both extension and retraction.
RESUMEN
TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.
Asunto(s)
Algoritmos , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Significant technical challenges have limited the study of extremophile cell biology. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius with unprecedented resolution. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.
RESUMEN
Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.
Asunto(s)
Adhesión Bacteriana , Agregación Celular , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Mutación , Neisseria meningitidis/fisiología , Proteínas Fimbrias/química , Regulación Bacteriana de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.
Asunto(s)
Adhesión Bacteriana , Membrana Celular/metabolismo , Nanofibras/química , Animales , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Liposomas , Ratones SCID , Modelos Biológicos , Nanofibras/ultraestructura , Neisseria meningitidis/metabolismo , Neisseria meningitidis/ultraestructura , HumectabilidadRESUMEN
Introducing concepts from soft matter physics and mechanics has largely contributed to our understanding of a variety of biological processes. In this review, we argue that this holds true for bacterial pathogenesis. We base this argument on three examples of bacterial pathogens and their interaction with host cells during infection: (i) Shigella flexneri exploits actin-dependent forces to come into close contact with epithelial cells prior to invasion of the epithelium; (ii) Neisseria meningitidis manipulates endothelial cells to resist shear stress during vascular colonization; (iii) bacterial toxins take advantage of the biophysical properties of the host cell plasma membrane to generate transcellular macroapertures in the vascular wall. Together, these examples show that a multidisciplinary approach integrating physics and biology is more necessary than ever to understand complex infectious phenomena. Moreover, this avenue of research will allow the exploration of general processes in cell biology, highlighted by pathogens, in the context of other non-communicable human diseases.
Asunto(s)
Infecciones Bacterianas , Animales , Células Endoteliales , Células Epiteliales , Epitelio , Humanos , Estrés FisiológicoRESUMEN
The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.
Asunto(s)
Endotelio Vascular/microbiología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Evasión Inmune , Modelos Moleculares , Neisseria meningitidis/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Adhesión Bacteriana , Línea Celular , Células Cultivadas , Secuencia Conservada , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/ultraestructura , Eliminación de Gen , Glicosilación , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/microbiología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/metabolismo , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/patología , Microscopía Electrónica de Transmisión , Neisseria meningitidis/inmunología , Neisseria meningitidis/ultraestructura , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Propiedades de SuperficieRESUMEN
Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-ß-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.