Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE.

Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling

AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.

Widespread Protection of RNA Cleavage Sites by a Riboswitch Aptamer that Folds as a Compact Obstacle to Scanning by RNase E.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

Membrane association and polar localization of the Legionella pneumophila T4SS DotO ATPase mediated by two nonredundant receptors.

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is a large, multisubunit complex that exports a vast array of substrates into eukaryotic host cells. DotO, a distant homolog of the T4ASS ATPase VirB4, associates with the bacterial inner membrane despite lacking hydrophobic transmembrane domains. Employing a genetic approach, we found DotO's membrane association is mediated by three inner-membrane Dot/Icm components, IcmT, and a combined DotJ-DotI complex (referred to as DotJI). Although deletion of icmT or dotJI individually does not affect DotO's membrane association, the simultaneous inactivation of all three genes results in increased amounts of soluble DotO. Nevertheless, deleting each receptor separately profoundly affects positioning of DotO, disrupting its link with the Dot/Icm complex at the bacterial poles, rendering the receptors nonredundant. Furthermore, a collection of dotO point mutants that we isolated established that DotO's N-terminal domain interacts with the membrane receptors and is involved in dimerization, whereas DotO's C-terminal ATPase domain primarily contributes to the protein's formation of oligomers. Modeling data revealed the complex interaction between DotO and its receptors is responsible for formation of DotO's unique "hexamer of dimers" configuration, which is a defining characteristic of VirB4 family members.

Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

The Sde phosphoribosyl-linked ubiquitin transferases protect the Legionella pneumophila vacuole from degradation by the host.

Legionella pneumophila grows intracellularly within the membrane-bound Legionella-containing vacuole (LCV) established by proteins translocated via the bacterial type IV secretion system (T4SS). The Sde family, one such group of translocated proteins, catalyzes phosphoribosyl-ubiquitin (pR-Ub) modification of target substrates. Mutational loss of the entire Sde family results in small defects in intracellular growth, making it difficult to identify a clear role for this posttranslational modification in supporting the intracellular lifestyle. Therefore, mutations that aggravate the loss of sde genes and caused intracellular growth defects were identified, providing a mechanistic connection between Sde function and vacuole biogenesis. These double mutants drove the formation of LCVs that showed vacuole disintegration within 2 h of bacterial contact. Sde proteins appeared critical for blocking access of membrane-disruptive early endosomal membrane material to the vacuole, as RNAi depletion of endosomal pathway components partially restored LCV integrity. The role of Sde proteins in preventing host degradation of the LCV was limited to the earliest stages of infection. The time that Sde proteins could prevent vacuole disruption, however, was extended by deletion of sidJ, which encodes a translocated protein that inactivates Sde protein active sites. These results indicate that Sde proteins act as temporally regulated vacuole guards during the establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments during the earliest steps of LCV biogenesis.

Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila.

Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

Molecular regulation of virulence in Legionella pneumophila.

Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

Tools and mechanisms of vacuolar escape leading to host egress in Legionella pneumophila infection: Emphasis on bacterial phospholipases.

The phenomenon of host cell escape exhibited by intracellular pathogens is a remarkably versatile occurrence, capable of unfolding through lytic or non-lytic pathways. Among these pathogens, the bacterium Legionella pneumophila stands out, having adopted a diverse spectrum of strategies to disengage from their host cells. A pivotal juncture that predates most of these host cell escape modalities is the initial escape from the intracellular compartment. This critical step is increasingly supported by evidence suggesting the involvement of several secreted pathogen effectors, including lytic proteins. In this intricate landscape, L. pneumophila emerges as a focal point for research, particularly concerning secreted phospholipases. While nestled within its replicative vacuole, the bacterium deftly employs both its type II (Lsp) and type IVB (Dot/Icm) secretion systems to convey phospholipases into either the phagosomal lumen or the host cell cytoplasm. Its repertoire encompasses numerous phospholipases A (PLA), including three enzymes-PlaA, PlaC, and PlaD-bearing the GDSL motif. Additionally, there are 11 patatin-like phospholipases A as well as PlaB. Furthermore, the bacterium harbors three extracellular phospholipases C (PLCs) and one phospholipase D. Within this comprehensive review, we undertake an exploration of the pivotal role played by phospholipases in the broader context of phagosomal and host cell egress. Moreover, we embark on a detailed journey to unravel the established and potential functions of the secreted phospholipases of L. pneumophila in orchestrating this indispensable process.

The LysR-type transcriptional regulator LelA co-regulates various effectors in different Legionella species.

The intracellular pathogen Legionella pneumophila translocates more than 300 effector proteins into its host cells. The expression levels of the genes encoding these effectors are orchestrated by an intricate regulatory network. Here, we introduce LelA, the first L. pneumophila LysR-type transcriptional regulator of effectors. Through bioinformatic and experimental analyses, we identified the LelA target regulatory element and demonstrated that it directly activates the expression of three L. pneumophila effectors (legL7, legL6, and legU1). We further found that the gene encoding LelA is positively regulated by the RpoS sigma factor, thus linking it to the known effector regulatory network. Examination of other species throughout the Legionella genus revealed that this regulatory element is found upstream of 34 genes encoding validated effectors, putative effectors, and hypothetical proteins. Moreover, ten of these genes were examined and found to be activated by the L. pneumophila LelA as well as by their orthologs in the corresponding species. LelA represents a novel type of Legionella effector regulator, which coordinates the expression of both adjacently and distantly located effector-encoding genes, thus forming small groups of co-regulated effectors.

Structure-function relationships underpin disulfide loop cleavage-dependent activation of Legionella pneumophila lysophospholipase A PlaA.

Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB-secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/ß-hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate-binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.

Membrane-dependent actin polymerization mediated by the Legionella pneumophila effector protein MavH.

L. pneumophila propagates in eukaryotic cells within a specialized niche, the Legionella-containing vacuole (LCV). The infection process is controlled by over 330 effector proteins delivered through the type IV secretion system. In this study, we report that the Legionella MavH effector localizes to endosomes and remodels host actin cytoskeleton in a phosphatidylinositol 3-phosphate (PI(3)P) dependent manner when ectopically expressed. We show that MavH recruits host actin capping protein (CP) and actin to the endosome via its CP-interacting (CPI) motif and WH2-like actin-binding domain, respectively. In vitro assays revealed that MavH stimulates actin assembly on PI(3)P-containing liposomes causing their tubulation. In addition, the recruitment of CP by MavH negatively regulates F-actin density at the membrane. We further show that, in L. pneumophila-infected cells, MavH appears around the LCV at the very early stage of infection and facilitates bacterium entry into the host. Together, our results reveal a novel mechanism of membrane tubulation induced by membrane-dependent actin polymerization catalyzed by MavH that contributes to the early stage of L. pneumophila infection by regulating host actin dynamics.

Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2-4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.

Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation.

The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease1-3. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown4-6. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages4,5. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca2+ concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

Ubiquitin and Legionella: From bench to bedside.

Legionella pneumophila, a Gram-negative intracellular bacterium, is one of the major causes of Legionnaires' disease, a specific type of atypical pneumonia. Despite intensive research efforts that elucidated many relevant structural, molecular and medical insights into Legionella's pathogenicity, Legionnaires' disease continues to present an ongoing public health concern. Legionella's virulence is based on its ability to simultaneously hijack multiple molecular pathways of the host cell to ensure its fast replication and dissemination. Legionella usurps the host ubiquitin system through multiple effector proteins, using the advantage of both conventional and unconventional (phosphoribosyl-linked) ubiquitination, thus providing optimal conditions for its replication. In this review, we summarize the current understanding of L. pneumophila from medical, biochemical and molecular perspectives. We describe the clinical disease presentation, its diagnostics and treatment, as well as host-pathogen interactions, with the emphasis on the ability of Legionella to target the host ubiquitin system upon infection. Furthermore, the interdisciplinary use of innovative technologies enables better insights into the pathogenesis of Legionnaires' disease and provides new opportunities for its treatment and prevention.

Requirement of phosphatidic acid binding for distribution of the bacterial protein Lpg1137 targeting syntaxin 17.

The gram-negative bacterium, Legionella pneumophila is known to manipulate the host cellular functions. L. pneumophila secretes bacterial proteins called Legionella effectors into the host cytosol that are necessary for these manipulations. The Legionella effector Lpg1137 was identified as a serine protease responsible for the degradation of syntaxin 17 (Stx17). However, how Lpg1137 specifically recognizes and degrades Stx17 remained unknown. Given that Stx17 is localized in the ER, mitochondria-associated membrane (MAM), and mitochondria, Lpg1137 likely distributes to these compartments to recognize Stx17. Here, we show that the C-terminal region of Lpg1137 binds to phosphatidic acid (PA), a MAM and mitochondria-enriched phospholipid, and that this binding is required for the correct intracellular distribution of Lpg1137. Two basic residues in the C-terminal region of Lpg1137 are required for PA binding and their mutation causes mislocalization of Lpg1137. This mutant also fails to degrade Stx17 while retaining protease activity. Taken together, our data reveal that Lpg1137 utilizes PA for its distribution to the membranous compartments in which Stx17 is localized.

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.