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Molecular architecture of bacterial type IV secretion systems [1]

['Michael J. Sheedlo', 'Department Of Pharmacology', 'University Of Minnesota', 'Minneapolis', 'Minnesota', 'United States Of America', 'Melanie D. Ohi', 'Department Of Cell', 'Developmental Biology', 'University Of Michigan']

Date: 2022-11 

Abstract 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.

Citation: Sheedlo MJ, Ohi MD, Lacy DB, Cover TL (2022) Molecular architecture of bacterial type IV secretion systems. PLoS Pathog 18(8): e1010720. https://doi.org/10.1371/journal.ppat.1010720 Editor: Christoph Dehio, University of Basel, SWITZERLAND Published: August 11, 2022 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by NIH AI118932 (TLC, MDO), CA116087 (TLC), AI039657 (TLC), AI164651 (MDO), and the Department of Veterans Affairs (I01BX004447)(TLC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Overview of bacterial type IV secretion systems Bacteria use multiple types of secretion systems to translocate molecules across the cell envelope [1–4]. Type IV secretion systems (T4SSs) are a functionally heterogeneous group of nanomachines that can deliver bacterial proteins and DNA into a diverse range of target cells, including eukaryotic cells (mammalian, plant, insect, and protozoa) and other bacteria [1,2,5–12]. T4SSs can be classified into 2 main groups: conjugation systems and effector translocation systems. Conjugation systems are specialized for transferring mobile genetic elements between bacteria [1,2,5–8]. Effector translocation systems deliver effector proteins, resulting in functional alterations in the target cells [1,2,5–8]. Some T4SSs (exemplified by the Legionella pneumophila Dot/Icm T4SS) can deliver several hundred different effector proteins into target cells [13,14]. T4SSs in Xanthomonas citri and Stenotrophomonas maltophilia deliver effector proteins into other bacteria, resulting in bacterial killing [15–17]. Some T4SSs can deliver bacterial DNA into eukaryotic cells [18,19]. For example, the Agrobacterium tumefaciens VirB/VirD4 T4SS translocates a T-DNA-relaxase complex and effector proteins into plant cells, resulting in tumor formation (crown gall disease) [20]. Other T4SSs have the capacity to export DNA or proteins into the extracellular environment (independent of contact with target cells) or import DNA [9]. T4SSs have been investigated mainly in gram-negative bacteria, but these secretion systems are also present in gram-positive bacteria and Archaea [6,9–12]. T4SS-mediated delivery of effector proteins into host cells contributes to the pathogenesis of numerous infections affecting humans [11]. For example, the Helicobacter pylori Cag T4SS has an important role in the pathogenesis of gastric cancer and peptic ulcer disease [21–23]. The L. pneumophila Dot/Icm T4SS is required for the pathogenesis of Legionnaire’s disease [14], and the Bordetella pertussis Ptl T4SS has a key role in the pathogenesis of pertussis (whooping cough) [24]. T4SSs also have important roles in the pathogenesis of infections caused by Bartonella, Brucella, Coxiella, Rickettsia, Ehrlichia, and Anaplasma [11]. The first insights into the structural organization of T4SSs came from studies of conjugation systems and the A. tumefaciens VirB/VirD4 system [7,25–27]. These prototype systems contain 12 components (designated VirB1-11 and VirD4) and are known as “minimized T4SSs” [8]. By convention, the term “minimized T4SS” refers to any T4SS in which the number of components is similar to the number in prototype VirB/VirD4 systems [8]. Some bacteria assemble “expanded T4SSs,” which are more complex than the minimized T4SSs [8]. Examples of expanded T4SSs include the H. pylori Cag T4SS, the L. pneumophila Dot/Icm T4SS, and the F plasmid-encoded T4SS. T4SSs in gram-negative bacteria have been classified into type IVa and type IVb subfamilies based on phylogenetic analysis of the sequences of components [12]. This phylogenetic classification does not mirror the classification of T4SSs into minimized and expanded categories. T4SSs in gram-positive bacteria, which typically have fewer components than T4SSs in gram-negative bacteria, have been classified in a type IVc subfamily [28,29]. Recent studies have provided important insights into the molecular architecture of T4SSs. In this review, we compare the macromolecular structures of T4SSs from multiple gram-negative bacterial species. We discuss features that are conserved among T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.

Methods for structural analysis of T4SSs An important first step in understanding the mechanisms of action of large molecular machines is defining their molecular architecture. Until recently, the only experimental approaches that consistently yielded high-resolution structures were X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which are not well-suited for analyzing the structures of large subassemblies. Technological developments in the field of cryo-electron microscopy (cryo-EM) now allow this technique to routinely determine structures at resolutions amenable to building molecular models directly into the EM density maps. Since EM does not require proteins to pack into crystals and can also be used with heterogenous and dynamic samples, this method can tackle problems that cannot be easily addressed by crystallography or NMR. New technological advances have also expanded the reach of cryo-EM to reveal structures of complexes in their native cellular environment. Cryo-electron tomography (cryo-ET) allows for the visualization of molecular complexes in the context of vitrified intact cells. When cryo-ET is combined with image analysis approaches such as sub-tomogram averaging, it is possible to attain sub-nanometer resolution of large cellular complexes. Synergy between X-ray crystallography, NMR, single-particle cryo-EM, and cryo-ET has led to an exciting period in the field, yielding important advances in our understanding of the molecular architecture of bacterial secretion systems [30,31].

Structural features of core complexes from minimized T4SSs High-resolution structures of subassemblies or individual components of minimized T4SSs have been generated by using either X-ray crystallography or single-particle cryo-EM methods. Early studies of protein–protein interactions among components of T4SSs suggested that several VirB proteins could assemble into one or more protein complexes [46]. Subsequently, 4 genes in the pKM101 conjugative plasmid [encoding TraN (VirB7), TraE (VirB8), TraO (VirB9), and TraF (VirB10)] were cloned and expressed, and a complex containing VirB7, VirB9, and VirB10 (but not VirB8) was isolated [47]. Cryo-EM analysis of the isolated particles revealed a barrel-shaped structure about 185 Å in height and diameter with 14-fold symmetry [47], corresponding to the pKM101 core complex visualized by cryo-ET [32]. Outer and inner layers (O- and I-layers) could be distinguished [47], similar to the organization visualized by cryo-ET (Fig 1) [32]. Chymotryptic cleavage of the purified core complex yielded the isolated O-layer, which contains the C-terminal portion of VirB9 (TraO), the C-terminal portion of VirB10 (TraF), and the full-length VirB7 (TraN) [48]. A crystal structure of the isolated O-layer showed that VirB10 is localized in the center, surrounded by VirB9 and VirB7. A central ring, composed of 14 two-helix bundles of VirB10 α-helices (designated as the “antenna region”), was proposed to form a channel through the outer membrane (Fig 2). In support of this hypothesis, an epitope tag inserted between VirB10 α-helices in the antenna region was detected extracellularly [48]. The I-layer is composed of N-terminal domains of VirB9 and VirB10 [49]. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Structure of core complexes from minimized T4SSs. The structures of core complexes from the pKM101 (PDB 3JQO) and X. citri (PDB 6GYB) T4SSs are shown as surface representations [48,52]. The O-layers are shown in blue, and the I-layer of the X. citri core complex is shown in gray. The asymmetric unit for each T4SS is indicated in yellow and consists of VirB7, VirB9, and VirB10. The structures of the asymmetric unit for each system are shown in the inset. T4SS, type IV secretion system. https://doi.org/10.1371/journal.ppat.1010720.g002 Further insights were provided by a study in which 10 genes in the R388 conjugative plasmid (virB1-10) were cloned and expressed [50]. Eight of the encoded proteins (VirB3-VirB10) assembled into a protein complex. EM analysis of the purified R388-encoded T4SS assembly revealed a core complex connected to a putative IMC by a thin stalk. It was proposed that VirB10 forms a connection between the core complex and the IMC. The IMC, predicted to be composed of the VirB4 ATPase, was organized into 2 side-by-side hexameric barrels [50]. Analysis of similar complexes containing VirD4 (an ATPase thought to be responsible for substrate recruitment) showed that VirD4 dimers are localized between the VirB4 ATPases [51]. Notably, the structural organization of the IMC visualized in the isolated T4SS particles [50] differed from that of the pKM101 IMC visualized by cryo-ET [32]. Despite this difference, both the cryo-ET analysis and analysis of isolated T4SS particles showed that the IMC has a molecular organization different from that of the core complex (6-fold and 14-fold symmetry, respectively) [32,50,51]. Like pKM101- and R388-encoded T4SS core complexes, core complexes within the X. citri and the A. tumefaciens VirB/VirD4 T4SSs are also composed of VirB7-, VirB9-, and VirB10-like proteins [52,53]. In contrast to the barrel-like shape of the pKM101 core complex, the X. citri core complex has a “flying saucer” shape (Fig 2) [52]. This feature of the X. citri T4SS is at least partially attributable to the presence of an additional domain in X. citri VirB7. High-resolution structural analysis of the X. citri core complex revealed features of the I-layer (composed of N-terminal portions of VirB9 and VirB10) that had not been resolved in the previous studies of conjugation systems [52]. Collectively, the analyses of these minimized T4SSs indicate that they all contain core complexes composed of VirB7, VirB9, and VirB10, organized into 2 ring-like layers (O-layer and I-layer) with 14-fold symmetry (Fig 2).

Structural analyses of inner membrane complexes and F pili In contrast to T4SS core complexes, which are relatively stable in detergent, intact IMCs have been difficult to purify, hindering high-resolution structural studies. Nevertheless, the structures of IMC subassemblies or individual IMC components have been successfully determined, using either cryo-EM or crystallization methods [63,64]. IMCs from minimized T4SSs are predicted to contain multiple ATPases (VirB4, VirB11, and VirD4) along with several other components (including VirB3, VirB5, VirB6, and VirB8) [50]. VirD4 plays a critical role in recruitment of effector proteins [7,8,65,66]. High-resolution structural models have been determined for the L. pneumophila Dot/Icm coupling complex, which functions to recognize and recruit effector proteins. The coupling complex contains DotL (a VirD4-like ATPase) and multiple additional proteins (including DotM, DotN, DotY, DotZ, lcmS, IcmW, and LvgA) [67–70]. The approximately 300 kDa mass of the complex suggests that it contains 1 copy of each component [69]. It is predicted that the approximately 300 kDa complexes assemble into larger hexameric subassemblies within the IMC. Conjugative pili have an important role in horizontal transfer of DNA among bacteria. F plasmid-encoded pili have been extensively studied and are characterized by an ability to dynamically extend and retract [8,33,40]. Cryo-EM studies showed that these structures are composed of thousands of copies of the TraA (VirB2) pilin bound to phospholipid [71,72]. The TraA-phospholipid building blocks of several T4SS pilus systems are organized as pentameric complexes in a helical assembly [71], whereas the building blocks of other T4SS pilus systems have a non-pentameric organization [72].

Conserved structural features of T4SSs Minimized T4SSs can translocate DNA and proteins across the cell envelope, and accordingly, many structural features of minimized T4SSs are conserved among the expanded T4SSs. The core complex is presumed to be one of the first structures assembled during T4SS biogenesis [35,38]. Proteins resembling VirB7, VirB9, and VirB10 are present in nearly all the T4SSs analyzed thus far and can be viewed as the fundamental building blocks for core complex assembly. VirB10-like proteins are predicted to span from the inner membrane to the outer membrane, and these proteins constitute the central portion of the core complex. A VirB10 “antenna region” is positioned at the apex of core complexes in all T4SSs analyzed thus far and is predicted to form an outer membrane channel [48]. The VirB10 antenna region is characterized by a “2-helix bundle ring system” [48]. The α-helical structure of this portion of VirB10 differs from the structures of most outer membrane proteins in gram-negative bacteria, which typically have a β-barrel architecture. The high conservation of C-terminal VirB10 sequences among T4SSs, the central localization of VirB10 within core complexes, and the apparent insertion of VirB10 into the outer membrane are consistent with a key role of VirB10 in core complex assembly and function. While VirB10 is thought to be essential for core complex assembly in many species [35], Legionella Dot/Icm core complexes can apparently assemble in the absence of VirB10 [38]. Conserved features of core complex organization include the presence of 2 layers (O- and I-layers) and the presence of radial spokes. The O-layers of core complexes in minimized systems contain VirB7 and C-terminal portions of VirB9 and VirB10, and the O-layers of expanded systems contain related components. The lipid components of the VirB7-like proteins are positioned close to the outer membrane [57]. The I-layers of core complexes in minimized systems contain N-terminal portions of VirB9 and VirB10 [52], and the compositions of the F-type T4SS I-layer, H. pylori PR, and Legionella PR mimic that of the I-layer in minimized systems. There are also similarities in the molecular architecture of the IMCs from multiple T4SSs, including 6-fold symmetry and the presence of multiple ATPases (along with other components). Symmetry mismatch is a striking feature of T4SSs. All the T4SSs examined thus far exhibit symmetry mismatch at the junction between the IMC and the core complex [32,33,35,37–39,50], and symmetry mismatch is also present within core complexes of expanded T4SSs [54–57,60,61]. Sites of symmetry mismatch are likely to provide regions of mobility or flexibility between adjacent layers. These sites might provide a “ratcheting” mechanism that keeps substrates moving in one direction. Thus far, it has been difficult to detect the proposed mobility between adjacent layers, and the mechanisms by which symmetry mismatch contribute to T4SS function remain poorly understood. Symmetry mismatch has been detected not only within T4SSs but also within several other types of bacterial secretion systems, including T2SSs, T3SSs (and related flagellar systems), and T6SSs [73–77]. The presence of symmetry mismatch within these diverse secretion systems suggests that it has an important functional significance.

Evolutionary strategies for T4SS diversification Recent studies of T4SSs from multiple bacterial species provide insight into the range of variation that exists in their molecular architecture. Incorporation of novel components is one of the recurring phenomena that can lead to variations in the architecture of T4SSs. For example, the core complexes of expanded T4SSs from H. pylori and L. pneumophila have diameters that are about twice the size of the diameters of core complexes in minimized systems, and this variation is attributed in part to the incorporation of species-specific components. The sequences and structures of the species-specific components in H. pylori are unrelated to those of the species-specific components in Legionella. Therefore, the genes encoding these components in the 2 species were probably acquired through independent horizontal transfer events. Interestingly, the structure of H. pylori Cag3 resembles that of H. pylori CagT (a VirB7 homolog), despite a lack of sequence relatedness between Cag3 and CagT [57]. The similarity in structures of Cag3 and CagT may be attributable to convergent evolution. At present, it is not known what benefits are conferred by the enlarged size of H. pylori and L. pneumophila T4SS core complex subassemblies. Current evidence suggests that T4SS effector proteins are translocated in an unfolded state [78,79], so the enlarged size is unlikely related to the size of the secreted effector proteins. Altering the stoichiometry of subassembly components is another mechanism leading to diversification of T4SS architecture. For example, the F-type T4SS core complex is assembled from the same 3 building blocks found in minimized systems, but additional copies of the VirB7 and VirB9 components (TraV and TraK) are observed within the asymmetric unit [54,55] (Fig 4). Similarly, within asymmetric units of the H. pylori Cag T4SS and L. pneumophila Dot/Icm core complexes, there are 2 copies of the VirB7 homologs (CagT and DotD, respectively) [57,61] (Fig 4). Thus, incorporation of an increased number of VirB7 and/or VirB9 components is a feature shared by core complexes in multiple expanded T4SSs. Among T4SS components that are conserved in T4SSs from multiple species, there is a high level of variation in the sequences and structures. Comparative analysis of VirB7, VirB9, and VirB10-like proteins in T4SSs from multiple species provides insight into the ways in which the sequences and structures of these proteins have diversified and how this affects T4SS architecture. One of the recurring features is incorporation of extra domains into these proteins, leading to variations in size. For example, VirB7 in the minimized T4SSs is a small protein (about 55 amino acids in length in A. tumefaciens), whereas VirB7-like proteins in H. pylori and Legionella are 280 and 163 amino acids in length, respectively. Similarly, VirB10- and VirB9-like proteins are much larger in H. pylori and Legionella T4SSs than in minimized systems. For example, CagY (VirB10 homolog) in H. pylori contains a large region characterized by amino acid repeat units [80], which is not present in VirB10 proteins in the minimized systems. There is also considerable variation in the symmetry of elements within core complexes from multiple T4SSs. For example, the O-layers of core complexes in minimized T4SSs have 14-fold symmetry, whereas core complexes from H. pylori, Legionella, and the F-type T4SS have 13-, 14-, 16-, 17-, or 18-fold symmetry in various regions. The functional significance of these variations and the structural determinants of symmetry organization are not yet known. Similarly, it is not known whether the exact geometry of symmetry mismatches is functionally important.

Specialized functions conferred by specific T4SS structural features T4SSs exhibit an extraordinary diversity of functions, and recent studies have revealed marked diversity in the molecular architectures of T4SSs. Presumably the specialized structural features of individual T4SSs are linked to their unique functions, but these relationships are not yet understood. As one approach for dissecting how specialized structural features contribute to specific functions, we can consider ways in which the structural organization of expanded T4SSs confers functional properties not exhibited by minimized T4SSs. For example, we speculate that the numerous species-specific components and structural complexity of the L. pneumophila Dot/Icm system contribute to its ability to secrete several hundred different effector proteins [13,14]. Similarly, we hypothesize that species-specific components and modifications of conserved T4SS components in the H. pylori Cag T4SS allow it to deliver multiple types of substrates (protein, DNA, lipopolysaccharide metabolites, and peptidoglycan) into host cells [21–23] and alter host cell physiology through translocation-independent mechanisms [81,82]. The organization of the F-type core complex (2 radially concentric rings exhibiting symmetry mismatch) presumably facilitates the processes required for pilus extension and retraction [40], as well as transitions between secretion of pilus components and secretion of plasmid DNA. Experimental studies, coupled with analyses of T4SSs from additional bacterial species, will be required to better understand the relationships between specialized structural and functional features of T4SSs.

Future prospects Studies conducted over the past decade have revealed both conserved features and variation in T4SS structural organization. For a more comprehensive view of T4SS diversity, it will be important to analyze T4SSs from additional species. Single-particle cryo-EM and crystallization methodology have been used to obtain high-resolution structural models of isolated T4SS subassemblies or individual proteins, and cryo-ET methods have been used to visualize T4SSs within intact bacteria. Future studies, focused on determining high-resolution structures of T4SS subassemblies in the context of intact membranes, will provide additional information about T4SS structure and function within a membrane environment. The studies conducted thus far have provided valuable insights into T4SS architecture and composition, but relatively little is known about the process of T4SS assembly, the functional roles of individual T4SS components, or the mechanisms of substrate translocation. In future studies, it will be important to define multiple conformational states of T4SSs, visualize intermediate stages in the processes by which T4SSs are assembled, and trace the path of effector proteins or other substrates through the assemblies. It will be important to define the functions of not only the conserved components of T4SSs but also the functions of species-specific components. Similarly, it will be important to determine the functional consequences of diversification in conserved T4SS components. Defining structural relationships between membrane-spanning T4SS components, pilus components, effector proteins, and other substrates at multiple stages of the translocation process will lead to a better understanding of the mechanisms of substrate translocation. We anticipate that such studies will reveal substantial mobility and conformational changes within the T4SS architecture, including mobility at sites of symmetry mismatch. Finally, we anticipate that the increasing wealth of T4SS structural data will help guide the development of agents that inhibit T4SS function, which might be useful in conjunction with traditional antibiotics for the treatment of bacterial infections.

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