细菌的IV型分泌系统

细菌的分泌系统与细菌的生存及致病性密切相关。细菌的分泌系统包括I-VI型,其中,IV型分泌系统是与细菌接合机制有关的一类分泌系统。IV型分泌系统不但可以转运DNA,还可以转运蛋白质及核糖核蛋白复合物等大分子物质,这点区别于其他几种分泌系统。IV型分泌系统介导基因水平转移,通过细菌间接合作用,传递抗性基因和毒力基因,有利于细菌进化;另一方面,IV型分泌系统转运效应蛋白质分子到宿主细胞,参与细菌致病。本文着重从IV型分泌系统几种主要类型的分泌机制等方面对IV型分泌系统进行概述。     

Novel type IV secretion system involved in propagation of genomic islands

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.     

Type IV secretion systems and their effectors in bacterial pathogenesis

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.     

Regulatory Networks of the T4SS Control: From Host Cell Sensing to the Biogenesis and the Activity during the Infection

Delivery of effectors, DNA or proteins, that hijack host cell processes to the benefit of bacteria is a mechanism widely used by bacterial pathogens. It is achieved by complex effector injection devices, the secretion systems, among which Type 4 Secretion Systems (T4SSs) play a key role in bacterial virulence of numerous animal and plant pathogens. Considerable progress has recently been made in the structure–function analyses of T4SSs. Nevertheless, the signals and processes that trigger machine assembly and activity during infection, as well as those involved in substrate recognition and transfer, are complex and still poorly understood. In this review, we aim at summarizing the last updates of the knowledge on signaling pathways that regulate the biogenesis and the activity of T4SSs in important bacterial pathogens.     

Euroconference on the Biology of Type IV Secretion Processes: bacterial gates into the outer world

Type IV secretion systems (T4SSs) mediate both protein and ssDNA secretion from a wide range of bacteria into virtually any cell type or into the milieu. It is this versatility that confers on them the ability to participate in many processes of bacterial life that imply communication with their environment. Type IV secretion systems are involved in horizontal DNA transfer to other bacteria and to plant cells, in DNA uptake from the milieu, in toxin secretion into the milieu, and in the injection of virulence factors into the eukaryotic host cell in a number of mammalian and plant pathogens. Recently, a EuroConference addressed the different aspects of the biology of these transmembrane multiprotein complexes, from the crystal structure of the individual components to the modification that the secreted substrates induce in the recipient cell. Significant progress has been made in the understanding of the molecular architecture and mechanism of secretion. The analysis of protein-protein interactions confirms the role of coupling proteins as substrate recruiters for the transporter. The VirB10 component of the complex has come up as a strong candidate for signal transducer. The wide range of effects on the recipient suggests that many effector proteins are secreted. New effector proteins are being identified for both plant and animal pathogens, as are their targets within the host cells. New T4SS members are being identified that perform novel roles, beyond DNA transfer and virulence, such as establishment of symbiotic processes. Our current knowledge of the Biology of Type IV Secretion Processes increases our ability to exploit them as biotechnological tools or to use them as new targets for inhibitors that could constitute a new generation of antimicrobials in the near future.     

Bacterial Type IV secretion systems: versatile virulence machines

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.     

Microreview: Type IV secretion systems: versatility and diversity in function

Type IV secretion systems (T4SSs) are large protein complexes which traverse the cell envelope of many bacteria. They contain a channel through which proteins or protein–DNA complexes can be translocated. This translocation is driven by a number of cytoplasmic ATPases which might energize large conformational changes in the translocation complex. The family of T4SSs is very versatile, shown by the great variety of functions among family members. Some T4SSs are used by pathogenic Gram‐negative bacteria to translocate a wide variety of virulence factors into the host cell. Other T4SSs are utilized to mediate horizontal gene transfer, an event that greatly facilitates the adaptation to environmental changes and is the basis for the spread of antibiotic resistance among bacteria. Here we review the recent advances in the characterization of the architecture and mechanism of substrate transfer in a few representative T4SSs with a particular focus on their diversity of structure and function.     

Mechanism and structure of the bacterial type IV secretion systems

The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the ‘archetypal’ A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Infection-associated type IV secretion systems of Bartonella and their diverse roles in host cell interaction

Type IV secretion systems (T4SSs) are transporters of Gram-negative bacteria that mediate interbacterial DNA transfer, and translocation of virulence factors into eukaryotic host cells. The α-proteobacterial genus Bartonella comprises arthropod-borne pathogens that colonize endothelial cells and erythrocytes of their mammalian reservoir hosts, thereby causing long-lasting intraerythrocytic infections. The deadly human pathogen Bartonella bacilliformis holds an isolated position in the Bartonella phylogeny as a sole representative of an ancestral lineage. All other species evolved in a separate 'modern' lineage by radial speciation and represent highly host-adapted pathogens of limited virulence potential. Unlike B. bacilliformis , the species of the modern lineage encode at least one of the closely related T4SSs, VirB/VirD4 or Vbh. These VirB-like T4SSs represent major host adaptability factors that contributed to the remarkable evolutionary success of the modern lineage. At the molecular level, the VirB/VirD4 T4SS was shown to translocate several effector proteins into endothelial cells that subvert cellular functions critical for establishing chronic infection. A third T4SS, Trw, is present in a sub-branch of the modern lineage. Trw does not translocate any known effectors, but produces multiple variant pilus subunits critically involved in the invasion of erythrocytes. The T4SSs laterally acquired by the bartonellae have thus adopted highly diverse functions during infection, highlighting their versatility as pathogenicity factors.     

Functional Analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae

Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons.     

The type IV secretion system of Sinorhizobium meliloti strain 1021 is required for conjugation but not for intracellular symbiosis

The type IV secretion system (T4SS) of the plant intracellular symbiont Sinorhizobium meliloti 1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.     

Structural Insights on Two Hypothetical Secretion Chaperones from <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv<Emphasis Type="Italic">. citri</Emphasis>

Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.     

Two pKM101‐encoded proteins,the pilus‐tip protein TraC and Pep,assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope‐spanning translocation channel, and those functioning in Gram‐negative species additionally elaborate an extracellular pilus to initiate donor‐recipient cell contacts. We report that pKM101, a self‐transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101‐encoded proteins, the pilus‐tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface‐displayed TraC and Pep are required for an efficient conjugative transfer, ‘extracellular complementation’ potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β‐barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS‐encoded, pilus‐independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.     

Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.     

Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Type IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five-protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.     

Protein interactions within and between two F-type type IV secretion systems

Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F-type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH-TM bacterial two-hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F-plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F-type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F-type T4SS-specific proteins. Furthermore, we demonstrate, for the first-time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F-type-specific proteins and we confirmed two of them by co-purification. The F-type-specific protein TraH_N was found to localize to the outer membrane and the presence of significant amounts of TraH_N in the outer membrane requires TraG_N.     

Molecular architecture of bacterial type IV secretion systems

Bacterial type IV secretion systems (T4SSs) are a versatile group of nanomachines that can horizontally transfer DNA through conjugation and deliver effector proteins into a wide range of target cells. The components of T4SSs in gram-negative bacteria are organized into several large subassemblies: an inner membrane complex, an outer membrane core complex, and, in some species, an extracellular pilus. Cryo-electron tomography has been used to define the structures of T4SSs in intact bacteria, and high-resolution structural models are now available for isolated core complexes from conjugation systems, the Xanthomonas citri T4SS, the Helicobacter pylori Cag T4SS, and the Legionella pneumophila Dot/Icm T4SS. In this review, we compare the molecular architectures of these T4SSs, focusing especially on the structures of core complexes. We discuss structural features that are shared by multiple T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.     

Exploring cargo transport mechanics in the type IV secretion systems

Type IV secretion systems (T4SSs) are used by various bacteria to deliver protein and DNA molecules to a wide range of target cells. These include systems that are directly involved in pathogenesis, such as the secretion of pertussis toxin by Bordetella pertussis into human cells and the delivery of single-stranded DNA (ssDNA) into plants by Agrobacterium. These complex systems are composed of proteins that span the bacterial cytoplasm. The Agrobacterium T4SS is composed of 12 virulence proteins and delivers its transferred ssDNA and several virulence protein substrates to a variety of eukaryotic cells. Recent studies on the Agrobacterium T4SS have revealed new information on the localization and structure of its proteins in the bacteria, the biochemical properties of its transport signal, the route of a DNA substrate through the secretion system, and the initial point of contact of the system with its host. These findings have expanded our knowledge and understanding of the still mostly obscure structure and function of the T4SSs.     

幽门螺杆菌Ⅳ型分泌系统(T4SS)研究进展

Ⅳ型分泌系统(T4SS)广泛存在于革兰阴性菌中,细菌可通过该系统将生物大分子或毒力因子等运输至靶细胞中并发挥相应功能。目前在H.pylori中已发现了至少三种T4SS,其中研究较为透彻的是cag致病岛(cagPAI)编码的cagT4SS系统,此外可塑区编码的tfs3系统和comB系统也有相关的报道。H.pylori的T4SS作为其与致病相关的重要结构已受到很多学者关注,对该菌T4SS系统的研究有助于进一步明确H.pylori的致病机制,并为临床诊断和治疗相关胃十二指肠疾病提供新的靶点。本文将对H.pylori的T4SS相关研究进展作一简要综述。