RESUMEN
Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.
Asunto(s)
Microscopía por Crioelectrón , Fimbrias Bacterianas , Sistemas de Secreción Tipo IV , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Modelos Moleculares , Conjugación Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Escherichia coli/genética , Conformación ProteicaRESUMEN
Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).
Asunto(s)
Proteínas Bacterianas , Stenotrophomonas maltophilia , Sistemas de Secreción Tipo IV , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/enzimología , Stenotrophomonas maltophilia/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Conformación ProteicaRESUMEN
The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from ΔcagT or ΔcagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY∆AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.
Asunto(s)
Helicobacter pylori , Sistemas de Secreción Tipo IV/química , PeriplasmaRESUMEN
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
Asunto(s)
Factor F , Sistemas de Secreción Tipo IV , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismo , Proteínas Fimbrias/genética , Plásmidos/genética , ADN Bacteriano , Proteínas Bacterianas/metabolismoRESUMEN
We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by theâ¯cagâ¯pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.
Asunto(s)
Adenosina Trifosfatasas , Proteínas Bacterianas , Helicobacter pylori , Helicobacter pylori/enzimología , Humanos , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/antagonistas & inhibidores , Diseño de Fármacos , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Modelos Moleculares , Sitios de Unión , Relación Estructura-Actividad , Línea Celular Tumoral , Interleucina-8/metabolismoRESUMEN
Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.
Asunto(s)
Fimbrias Bacterianas , Sistemas de Secreción Tipo IV , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Fimbrias Bacterianas/metabolismo , Bacterias/genética , Bacterias/metabolismo , Bacterias Gramnegativas/metabolismo , ADN , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
The recently published high-resolution R388 T4SS structure provides exciting new details about the complete complex of T4SS, including the components making up the stalk and arches, numerous symmetry mismatches between regions of the complex, and an intriguing interpretation of the closed stalk and radial symmetry of the inner membrane complex, which is related to pilus biogenesis assembly. However, there are a few unidentified densities in the electron microscopy map and portions of the identified component sequences for which the structure is not yet known. It is also unclear how well this minimized DNA-transporting T4SS predicts the structure of other T4SSs, such as expanded systems and those that transport proteins rather than DNA. In this review, we evaluate what can be inferred from the recent high-resolution structure of the R388 T4SS with respect to the Cag and Dot/Icm systems. These systems were selected because, given what is currently known about these systems, we expect them to present most structural differences compared to the R388 T4SS structure. Furthermore, we discuss bacterial physiology and diversity, the T4SS structures and their variations between different bacterial species. These insights may prove beneficial for researchers who elucidate the structure and functions of T4SS in different bacterial species.
Asunto(s)
ADN , Sistemas de Secreción Tipo IV , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo , Transporte Biológico , Proteínas Bacterianas/genéticaRESUMEN
Conjugative transfer is mediated by specialized type IV secretion systems (T4SSs). However, their architecture and mode of function remain poorly defined in Gram-positives. In this issue of Structure, Jäger et al. reveal an exclusive assembly of PrgL and illustrate the importance of its structural organization in pCF10 conjugative transfer.
Asunto(s)
Proteínas Bacterianas , Sistemas de Secreción Tipo IV , Proteínas Bacterianas/química , Plásmidos , Sistemas de Secreción Tipo IV/químicaRESUMEN
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
Multidrug-resistant bacteria pose serious problems in hospital-acquired infections (HAIs). Most antibiotic resistance genes are acquired via conjugative gene transfer, mediated by type 4 secretion systems (T4SS). Although most multidrug-resistant bacteria responsible for HAIs are of Gram-positive origin, with enterococci being major contributors, mostly Gram-negative T4SSs have been characterized. Here, we describe the structure and organization of PrgL, a core protein of the T4SS channel, encoded by the pCF10 plasmid from Enterococcus faecalis. The structure of PrgL displays similarity to VirB8 proteins of Gram-negative T4SSs. In vitro experiments show that the soluble domain alone is enough to drive both dimerization and dodecamerization, with a dimerization interface that differs from all other known VirB8-like proteins. In vivo experiments verify the importance of PrgL dimerization. Our findings provide insight into the molecular building blocks of Gram-positive T4SS, highlighting similarities but also unique features in PrgL compared to other VirB8-like proteins.
Asunto(s)
Proteínas Bacterianas , Sistemas de Secreción Tipo IV , Proteínas Bacterianas/química , Dimerización , Plásmidos , Conformación Proteica , Sistemas de Secreción Tipo IV/químicaRESUMEN
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
Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of â¼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Escherichia coli/química , Sistemas de Secreción Tipo IV/antagonistas & inhibidores , Sistemas de Secreción Tipo IV/química , Xanthomonas/química , Proteínas Bacterianas/genética , Escherichia coli/genética , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Relación Estructura-Actividad , Sistemas de Secreción Tipo IV/genética , Xanthomonas/genéticaRESUMEN
Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose "reader" proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.
Asunto(s)
Legionella pneumophila/crecimiento & desarrollo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/enzimología , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Células THP-1 , UbiquitinaciónRESUMEN
Conjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/metabolismo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , ADN Bacteriano , Escherichia coli/genética , Fimbrias Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Plásmidos , Sistemas de Secreción Tipo IV/genéticaRESUMEN
Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as "minimized" or "expanded" based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens transfer DNA (T-DNA) and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1 to VirB11 and VirD4. We visualized the pKM101-encoded T4SS in its native cellular context by in situ cryo-electron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by in situ CryoET but differs strikingly from the IMC of the purified T4SSR388, whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers of dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer. IMPORTANCE Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryo-electron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely related T4SS analyzed in vitro by single-particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for the development of intervention strategies aimed at suppressing the action of T4SS nanomachines.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/genética , Agrobacterium tumefaciens/enzimología , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/metabolismo , Conjugación Genética , Tomografía con Microscopio Electrónico/métodos , Metabolismo Energético , Escherichia coli/genética , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Microscopía por Crioelectrón/métodos , Legionella pneumophila/química , Proteínas Bacterianas/química , Conformación Proteica , Especificidad de la Especie , Sistemas de Secreción Tipo IV/químicaRESUMEN
Many gram-negative bacteria use type IV secretion systems to deliver effector molecules to a wide range of target cells. These substrate proteins, which are called type IV secreted effectors (T4SE), manipulate host cell processes during infection, often resulting in severe diseases or even death of the host. Therefore, identification of putative T4SEs has become a very active research topic in bioinformatics due to its vital roles in understanding host-pathogen interactions. PSI-BLAST profiles have been experimentally validated to provide important and discriminatory evolutionary information for various protein classification tasks. In the present study, an accurate computational predictor termed iT4SE-EP was developed for identifying T4SEs by extracting evolutionary features from the position-specific scoring matrix and the position-specific frequency matrix profiles. First, four types of encoding strategies were designed to transform protein sequences into fixed-length feature vectors based on the two profiles. Then, the feature selection technique based on the random forest algorithm was utilized to reduce redundant or irrelevant features without much loss of information. Finally, the optimal features were input into a support vector machine classifier to carry out the prediction of T4SEs. Our experimental results demonstrated that iT4SE-EP outperformed most of existing methods based on the independent dataset test.
Asunto(s)
Evolución Molecular , Bacterias Gramnegativas/genética , Interacciones Huésped-Patógeno/genética , Sistemas de Secreción Tipo IV/genética , Secuencia de Aminoácidos/genética , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/genética , Infecciones Bacterianas/microbiología , Biología Computacional , Bacterias Gramnegativas/patogenicidad , Humanos , Sistemas de Secreción Tipo IV/químicaRESUMEN
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.