RESUMEN
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Sistemas de Secreción Tipo IV , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Conjugación Genética , ADN/genética , Evolución Molecular , Fimbrias Bacterianas/metabolismo , Plásmidos/genética , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCCF) of a T4SS encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCCF consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB ß-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SSF function. This work identifies OMCCF structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Factor F/metabolismo , Sistemas de Secreción Tipo IV/química , Membrana Celular/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
Asunto(s)
Conjugación Genética , Fimbrias Bacterianas/fisiología , Bacterias Gramnegativas/química , Bacterias Gramnegativas/fisiología , Sistemas de Translocación de Proteínas/metabolismo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/fisiología , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/fisiología , Secuencias de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Microscopía por Crioelectrón , Bacterias Gramnegativas/ultraestructura , Infecciones por Bacterias Gramnegativas/microbiología , Helicobacter pylori/química , Helicobacter pylori/fisiología , Humanos , Legionella pneumophila/química , Legionella pneumophila/fisiología , Simulación del Acoplamiento Molecular , Sistemas de Translocación de Proteínas/química , Sistemas de Translocación de Proteínas/ultraestructura , Relación Estructura-Actividad , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires' Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Legionella pneumophila/química , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/ultraestructura , Microscopía por Crioelectrón , Conformación ProteicaRESUMEN
The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing five proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Helicobacter pylori/química , Sistemas de Secreción Tipo IV/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Microscopía por Crioelectrón , Modelos Moleculares , Conformación Proteica , Especificidad de la Especie , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/clasificaciónRESUMEN
Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane.IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.
Asunto(s)
Adenosina Trifosfatasas/ultraestructura , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Legionella pneumophila/metabolismo , Sistemas de Secreción Tipo IV/ultraestructura , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/ultraestructura , Modelos Moleculares , Conformación Proteica , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Bacterial type IV secretion systems (T4SSs) are molecular machines that can mediate interbacterial DNA transfer through conjugation and delivery of effector molecules into host cells. The Helicobacter pylori Cag T4SS translocates CagA, a bacterial oncoprotein, into gastric cells, contributing to gastric cancer pathogenesis. We report the structure of a membrane-spanning Cag T4SS assembly, which we describe as three sub-assemblies: a 14-fold symmetric outer membrane core complex (OMCC), 17-fold symmetric periplasmic ring complex (PRC), and central stalk. Features that differ markedly from those of prototypical T4SSs include an expanded OMCC and unexpected symmetry mismatch between the OMCC and PRC. This structure is one of the largest bacterial secretion system assemblies ever reported and illustrates the remarkable structural diversity that exists among bacterial T4SSs.
Asunto(s)
Helicobacter pylori/metabolismo , Sistemas de Secreción Tipo IV/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Modelos Moleculares , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagß and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The CagT4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the CagT4SS aligns structurally much more closely to the Dot/IcmT4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.
Asunto(s)
Proteínas Bacterianas/genética , Helicobacter pylori/genética , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/ultraestructura , Antígenos Bacterianos/genética , Microscopía por Crioelectrón , Islas Genómicas , HumanosRESUMEN
Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.
Asunto(s)
Legionella pneumophila/química , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/ultraestructura , Polaridad Celular , Tomografía con Microscopio Electrónico , Legionella pneumophila/citología , Legionella pneumophila/genética , Legionella pneumophila/ultraestructura , Microscopía Fluorescente , Mutación , Periplasma/química , Periplasma/ultraestructura , Multimerización de Proteína , Sistemas de Secreción Tipo IV/químicaRESUMEN
Type 4a pili (T4aP) are long, thin and dynamic fibres displayed on the surface of diverse bacteria promoting adherence, motility and transport functions. Genomes of many Enterobacteriaceae contain conserved gene clusters encoding putative T4aP assembly systems. However, their expression has been observed only in few strains including Enterohaemorrhagic Escherichia coli (EHEC) and their inducers remain unknown. Here we used EHEC genomic DNA as a template to amplify and assemble an artificial operon composed of four gene clusters encoding 13 pilus assembly proteins. Controlled expressions of this operon in nonpathogenic E. coli strains led to efficient assembly of T4aP composed of the major pilin PpdD, as shown by shearing assays and immunofluorescence microscopy. When compared with PpdD pili assembled in a heterologous Klebsiella T2SS type 2 secretion system (T2SS) by using cryo-electron microscopy (cryoEM), these pili showed indistinguishable helical parameters, emphasizing that major pilins are the principal determinants of the fibre structure. Bacterial two-hybrid analysis identified several interactions of PpdD with T4aP assembly proteins, and with components of the T2SS that allow for heterologous fibre assembly. These studies lay ground for further characterization of the T4aP structure, function and biogenesis in enterobacteria.
Asunto(s)
Escherichia coli Enterohemorrágica/metabolismo , Fimbrias Bacterianas/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Microscopía por Crioelectrón , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/ultraestructura , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/ultraestructura , Klebsiella/genética , Klebsiella/metabolismo , Microscopía Fluorescente , Unión Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos Híbridos , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche.
Asunto(s)
Helicobacter pylori/metabolismo , Sistemas de Secreción Tipo IV/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Microscopía por Crioelectrón , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Humanos , Legionella/metabolismo , Sistemas de Secreción Tipo IV/ultraestructuraRESUMEN
Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.
Asunto(s)
Brucella abortus/ultraestructura , Brucella melitensis/ultraestructura , Retículo Endoplásmico/ultraestructura , Vacuolas/ultraestructura , Animales , Brucella abortus/patogenicidad , Brucella melitensis/patogenicidad , Citoplasma/microbiología , Retículo Endoplásmico/microbiología , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Ratones , Microscopía Electrónica de Rastreo , Trofoblastos/microbiología , Trofoblastos/ultraestructura , Sistemas de Secreción Tipo IV/ultraestructura , Vacuolas/microbiologíaRESUMEN
Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.
Asunto(s)
Agrobacterium tumefaciens/ultraestructura , Sustancias Macromoleculares/aislamiento & purificación , Sustancias Macromoleculares/ultraestructura , Sistemas de Secreción Tipo IV/aislamiento & purificación , Sistemas de Secreción Tipo IV/ultraestructura , Agrobacterium tumefaciens/genética , Conjugación Genética , Microscopía Electrónica de Transmisión , Mapas de Interacción de ProteínasRESUMEN
Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Legionella pneumophila/química , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Regulación Bacteriana de la Expresión Génica , Legionella pneumophila/genética , Legionella pneumophila/patogenicidad , Legionella pneumophila/ultraestructura , PlásmidosRESUMEN
UNLABELLED: Bacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this report, we describe the isolation and analysis of a membrane-spanning core complex from the Helicobacter pylori cag T4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pylori proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to H. pylori. Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by Δcag3 or ΔcagT mutants retain the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirm that Cag3 is a peripheral component of the complex. The cag T4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the H. pylori cag T4SS that are optimized for function in the human gastric mucosal environment. IMPORTANCE: Type IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pylori is an important cause of stomach cancer, and the H. pylori T4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a "bacterial oncoprotein") into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pylori T4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system.
Asunto(s)
Helicobacter pylori/química , Helicobacter pylori/genética , Sustancias Macromoleculares/ultraestructura , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/ultraestructura , Humanos , Inmunohistoquímica , Microscopía ElectrónicaRESUMEN
Type IV secretion systems (T4SSs) are large multisubunit translocons, found in both gram-negative and gram-positive bacteria and in some archaea. These systems transport a diverse array of substrates from DNA and protein-DNA complexes to proteins, and play fundamental roles in both bacterial pathogenesis and bacterial adaptation to the cellular milieu in which bacteria live. This review describes the various biochemical and structural advances made toward understanding the biogenesis, architecture, and function of T4SSs.
Asunto(s)
Bacterias/metabolismo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/ultraestructura , Bacterias/química , Bacterias/clasificación , Factor F/genética , Microscopía ElectrónicaRESUMEN
Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the "pilus". Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface(. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.