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Biochem. J. (2010) 425, 475–488 (Printed in Great Britain) 475 doi:10.1042/BJ20091518 REVIEW ARTICLE Two-step and one-step secretion mechanisms in Gram-negative bacteria: contrasting the type IV secretion system and the chaperone-usher pathway of pilus biogenesis Ana Toste RÊGO1 , Vidya CHANDRAN1 and Gabriel WAKSMAN1,2 Institute of Structural and Molecular Biology, Birkbeck and University College London, Malet Street, London, WC1E 7HX, U.K. Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the onestep mechanism, whereby substrates are translocated directly from the bacterial cytoplasm to the extracellular medium or into the eukaryotic target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate transport across the outer membrane and released outside the bacterial cell. The present review provides an example for each of these two classes of secretion systems and contrasts the various solutions evolved to secrete substrates. INTRODUCTION Two main mechanisms for transport operate in the various secretion systems described above: one-step or Sec-independent compared with two-step or Sec-dependent transport mechanisms. The T2SS, T5SS and the CU systems lack an inner membrane transporter that can transfer substrates across the inner membrane in response to ATP. For the first step of transfer across the inner membrane, these systems require either the general secretory pathway, also called Sec translocon, or the Tat pathway in the case of some T2SS substrates. Specific secretion system machineries are then used for transport of substrates from the periplasm across the outer membrane. These systems thus utilize a two-step or Sec-dependent mechanism for translocation. On the other hand, T1SS, T3SS, T4SS and T6SS all utilize a simultaneous onestep translocation of substrates across both membranes. These are thus Sec-independent secretion pathways. The CU pathway is a very simple two-step translocation pathway requiring just two specialized proteins: the periplasmic chaperone and an outer membrane usher [10,11]. The T4SS, on the other hand, is a complex assembly of 12 or more proteins spanning across both the membranes with cytoplasmic ATPases and inner and outer membrane proteins assisting substrate secretion and pilus biogenesis [12]. The CU pathway and T4SS are thus examples of two types of secretion nanomachines, one utilizing a two-step mechanism for secretion (the CU pathway) and the other a onestep one (T4SS). In the present review, we use these two systems to compare and contrast the architectures and secretion mechanisms of these two classes of secretion systems. Other secretion systems could have been chosen for the same purpose, but describing them all would have made for a large and unwieldy review, the length of which was beyond the limits imposed for reviews published in this journal. Instead, the authors have opted for a review comparing Gram-negative bacteria have evolved several simple to complex machineries for transport of substrates across their cell membrane in response to various environmental cues. These machineries, known as secretion systems, are essential for bacterial virulence and are used by bacteria to acquire nutrients, transport various proteins, nucleic acids or toxins or assemble cell-surface organelles such as pili or fimbriae. Transport has to occur across both the membranes with the periplasmic space enclosed within. To achieve this, bacteria have evolved at least seven different secretion systems classified as type I (T1SS) to type VI (T6SS) secretion systems and the CU (chaperone-usher) system [1,2]. T1SS is a tripartite system made up of an inner membrane ABC (ATP-binding cassette) transporter, an adaptor protein bridging the two membrane components and an outer membrane pore [3]. The T2SS (type II secretion system) spans both the membranes but require the Sec or Tat pathway for transporting substrates across the inner membrane [4]. The T3SS (type III secretion system) or injectisome forms large supramolecular structures spanning both membranes and is structurally and evolutionarily related to bacterial flagella [5]. The T4SS (type IV secretion system) also forms large assemblies spanning both membranes and is evolutionarily related to the conjugation machinery [6,7]. The T5SS (type V secretion systems) are autotransporters or twopartner secretion systems assembled only in the outer membrane [8]. The T6SS is a recently discovered multi-protein system still largely uncharacterized [2]. Finally, the CU system is a simple system assembled in the outer membrane and responsible for assembly and secretion of virulence surface organelles [9,10]. Key words: chaperone-usher, protein secretion, structural biology, type IV secretion, virulence factor. Abbreviations used: AAD, all-α-helical domain; CU, chaperone-usher; DSC, donor-strand complementation; DSE, donor-strand exchange; EM, electron microscopy; I-layer, inner layer; NBD, nucleotide-binding domain; Nte, N-terminal extension; O-layer, outer layer; T1SS, T2SS etc., type I secretion system, type II secretion system etc; TM, transmembrane. 1 These authors contributed equally to the present review. 2 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2010 Biochemical Society 476 A.T. Rêgo, V. Chandran and G. Waksman the one- and two-step secretion systems they study, i.e. the CU pathway and the T4SS. CU SECRETION SYSTEM The CU system is responsible for the assembly and secretion of pili (also called fimbria), multisubunit cell surface appendages present in a wide range of pathogenic Gram-negative bacteria (e.g. Escherichia coli, Salmonella enterica, Yersinia species, Klebsiella pneumoniae, Pseudomonas aeruginosa etc.) [13]. CU pili are virulence factors employed by bacteria for host recognition and attachment, invasion and biofilm formation. CU pili are polymers of protein subunits. Two specialized proteins are required for CU pilus assembly: a periplasmic chaperone that stabilizes pilus subunits and primes them for subsequent polymerization into a pilus [14], and an outer membrane usher that works as the assembly platform where the chaperone subunit complexes are recruited, the subunits are polymerized and translocated for presentation at the cell surface [10,11]. CU pilus morphology varies from linear thick rods to more flexible thin structures. Phylogenetic analysis of usher sequences classified them into six major clades: α, β, γ (subdivided into γ 1 , γ 2 , γ 3 and γ 4 ), κ, π and σ fimbriae [15]. Rod-like fimbria, also called ‘typical’ fimbria (found in α, γ and π clades) are one of the best studied morphology types of the CU family, mainly through extensive studies of P and Type 1 pili (π and γ 1 fimbrial clades respectively) in UPEC (uropathogenic E. coli). Architecture of P and Type 1 CU pili CU fimbriae are typically encoded in individual gene clusters, where all subunits in addition to the chaperone and usher are encoded. The number of subunits varies from system to system. For instance, the Type 1 pili cluster codes for four known subunits (Fim A, F, G and H), whereas the P pili cluster codes for six known subunits (Pap A, H, K, E, F and G). In both systems, these subunits are arranged into two sub-assemblies: the pilus ‘rod’ and the ‘tip fibrillum’ (Figure 1). The rod is a long thick rigid structure with a ∼ 6.8 nm diameter made up of repeating subunits of PapA or FimA (>1000 copies), in P and Type 1 pili respectively. These repeating subunits form a right-handed helical structure with 3.3 subunits per turn [16,17]. The tip fibrillum of the P pilus is a thin and flexible structure with a ∼ 2 nm diameter, made of repeating PapE subunits (5–10 subunits) arranged in an open helical form and connected to the rod by a copy of the adaptor subunit PapK (Figure 1). A second single copy adaptor subunit PapF bridges the distal end specialized adhesin subunit PapG with the PapE polymer [18]. PapG has been shown to specifically recognize the globo-series of glycolipids present in the human kidney, and PapG has been shown to be a major virulence factor in kidney infections [19,20]. In Type 1 pili, the linear tip fibrillum is simply formed by one copy of each of the subunits FimF, FimG and the adhesin FimH [21,22] (Figure 1B). For that reason, Type 1 pili form shorter tip fibres. FimH recognizes mannose residues present in the human bladder [23] and Type 1 pili have been shown to be responsible for bladder infections (cystisis), as well as being implicated in attachment to abiotic surfaces in a model biofilm system [24,25]. The P pilus rod is terminated at the base by the PapH subunit that anchors the pilus structure to the cell wall [26,27] (Figure 1A). No similar subunit has yet been found in the Type 1 pili. c The Authors Journal compilation c 2010 Biochemical Society Figure 1 General diagram of the CU mechanism (A) Diagram of the P pilus representing all the subunits involved at the outer membrane. Each subunit is represented by oval shapes with a distinct colour and a letter; for example PapG is labelled G, Pap F is labelled F, and so forth. (B) Diagram of Type 1 pili with the same colour scheme as in (A). The pilus biogenesis is initiated by the export of the unfolded pilus subunits to the periplasm via the Sec translocation machinery, schematically represented in the inner membrane [155]. The periplasmic chaperone then captures subunits at the exit of the Sec translocon and assists in their folding and targeting to the outer membrane dimeric usher where one of the pores is activated. Once at the activated pore, subunits polymerize in an ordered sequence until pili completion. The extracellular, outer membrane, periplasmic space, inner membrane and cytoplasm are labelled E, OM, P, IM and C respectively. Assembly of CU pilus The initial step of the pilus biogenesis is the export of the pilus subunits to the periplasm via the Sec translocation machinery (Figure 1) (Sec-mediated transport through the inner membrane is not within the scope of the present review. For excellent reviews on this aspect, we refer the reader to [28,29]). At the exit of the Sec machinery, each subunit interacts with a periplasmic chaperone (PapD and FimC for P and Type 1 pilus subunits respectively) for proper folding and guidance to the outer membrane usher. In the absence of the chaperone, subunits aggregate and DegP protease transcription is activated, promoting subunit degradation [14,30,31]. Two-step versus one-step secretion systems Figure 2 477 DSC and DSE (A) DSC. Left panel: topology diagram of PapK (yellow) showing the PapD G1 (red) in the gap between strand A1 and F. Middle panel: structure of the PapD–PapK complex (PDB: 1PDK). PapD and PapK are in ribbon and surface representations respectively, colour-coded red and yellow respectively. The location of the P1 to P4 pockets in the subunit’s groove as well as that of the conserved chaperone residues co-ordinating the subunit’s C-terminus (Arg8 and Lys112 ) area are indicated. Right panel: close-up view of the P5 pocket of PapK. (B) DSE. Left panel: topology diagram of PapF (in green) bound to PapD (in red). Middle panel: topology diagram of the same subunit (in green) bound to Nte of PapK (in yellow). Right panel: surface representation of PapF bound to the Nte of PapK (PDB: 1N12). The location of the P2 to P5 pockets is shown. (C) The PapD–PapH structure (PDB: 2J2Z). Left panel: PapD and PapH are in surface and ribbon representations respectively, colour-coded in red and grey respectively. The location of the P2 to P5 pockets is shown. Right panel: a close-up view illustrating the fact that PapH does not have a P5 pocket. Mechanism of chaperone function: donor strand complementation CU chaperones have a conserved boomerang-like structure, composed of two Ig-like domains [32,33]. They possess two conserved residues essential for forming complexes with the subunits (Figure 2A). These residues are located between the two domains of the chaperone. In the PapD chaperone, these residues are Arg8 and Lys112 , and other chaperones have homologous residues in the same position [33,34]. Mutations in these residues abolishes pilus biogenesis [33]. Formation of chaperone– subunit complexes is also dependent upon a series of alternating hydrophobic surface residues (P1 to P4 residues) located in the last β-strand of the chaperone’s N-terminal domain (strand G1 ) (Figure 2A). Subunits share an incomplete Ig-like structure [35,36] with only six of the seven characteristic β-strands (βA to βF) present in the fold (Figures 2A and 2B). The C-terminal β-strand (βG) is entirely missing. As a result, a hydrophobic groove in the structure is created where the missing secondary structure should be. Because of the missing strand, subunits are unstable and unable to fold correctly on their own. Folding and stabilization of subunits is afforded by the insertion of the chaperone’s G1 strand into the subunit’s hydrophobic groove and interaction of the conserved arginine and lysine residues with the C-terminal c The Authors Journal compilation c 2010 Biochemical Society 478 A.T. Rêgo, V. Chandran and G. Waksman carboxylic group of the subunit’s strand F (Figure 2A). Insertion of the G1 strand results in its P1 to P4 residues occupying the P1 to P4 sites/pockets of the subunit’s groove (Figure 2A). Thus the chaperone ‘donates’ in trans the missing secondary structure: this mechanism of chaperone-mediated subunit folding and stabilization is termed DSC (donor-strand complementation) [14,30,35–37]. The resulting complemented fold, however, is not a canonical Ig-fold, since the chaperone G1 strand runs parallel to the subunits F strand instead of anti-parallel [38]. Mechanism of subunit polymerization: donor-strand exchange Every pilus subunit possesses a 10–20 residue long disordered Nte (N-terminal extension), which, similar to the G1 β-strand of the chaperone, contains alternating hydrophobic residues. This Nte was shown not to be part of the Ig-like fold [36]. During the course of polymerization, the Nte of the incoming subunit displaces the G1 donor β-strand of the chaperone, complementing the Ig-like fold of the previous subunit assembled in the pili [39] (Figure 2B). This mechanism of subunit polymerization is called ‘donor-strand exchange’ (DSE) [35,36,39,40]. In the subunit groove, the chaperone G1 strand hydrophobic residues occupy the P1 to P4 sites/pockets, as mentioned above, whereas after DSE the incoming Nte hydrophobic residues (termed P2 to P5 residues) occupy sites/pockets P2 to P5 of the groove (Figures 2A and 2B). The subunit fold is now a canonical Igfold, since the Nte strand runs anti-parallel to the subunit’s F strand, thus in a more energetically favourable conformation. The reaction by which the chaperone G1 strand is substituted by the incoming subunit Nte in the groove proceeds via a zipin-zip-out mechanism, whereby the process of DSE is initiated by the insertion of the P5 residue of the incoming subunit’s Nte into the P5 pocket of the previously assembled subunit groove [41,42] (see Figure 2A, right panel for the P5 pocket). DSE progresses with the new Nte gradually displacing the chaperone’s G1 strand step by step from P5 to P1 [42] (Figure 2B). The receiving subunit structure then transitions from a chaperonestabilized semi-unfolded state of high-energy to a folded state of lower energy when in interaction with an other subunit [43]: it is believed that it is the folding energy released upon DSE that drives subunit polymerization [43,44]. No other external source of energy is required to drive pilus polymerization. Termination of pilus biogenesis happens upon incorporation of the subunit PapH in the growing pilus. This happens because PapH is unable to undergo DSE since it lacks a P5 pocket [27] (Figure 2C). No mechanism for type 1 pilus termination has been found yet as a PapH-like termination subunit is unknown in that pilus system. The usher assembly platform In vivo DSE occurs at the outer membrane usher [44]. DSE is observed in vitro and in the absence of the usher, but proceeds only at very slow rates [45]. Rapid fibre formation, even in vitro, requires the presence of the usher. Thus the usher acts as a catalyst of DSE [46]. Outer membrane ushers are ∼ 90 kDa proteins with four distinct functional domains: an N-terminal periplasmic domain with ∼ 125 residues, a large translocation pore domain with ∼ 500 residues that is interrupted by a soluble plug domain of ∼ 110 residues and a C-terminal periplasmic domain with ∼ 170 residues [47,48] (Figure 3A). The N-terminal domain binds chaperone–subunit complexes [49,50] with binding affinities for each of the complexes decreasing in an order reflecting the natural order of subunits c The Authors Journal compilation c 2010 Biochemical Society in the pilus, a process that is believed to be responsible, in part, for subunit ordering. Structural and biophysical studies of the N-terminal domain of the FimD usher bound to both FimC–FimH and FimC–FimF showed that a flexible tail at the N-terminus (residues 1–24) was responsible for 40%–50 % of the binding interface and that tail was directly interacting with the subunit (FimH or FimF) (Figure 3B). Deletion mutants of residues 1–24 abolished chaperone–subunit interaction with the usher [49,51]. Similarly, for the PapC N-terminal domain, deletion of residues 1–11 abolished targeting of the usher to chaperone– subunit complexes [50]. Thus the tail of the N-terminal domain seems to be responsible for subunit-specific interactions. In vitro, the N-terminal domain doesn’t seem to be fundamental for pilus biogenesis, which may suggest that its role in vivo may be to capture chaperone–subunit complexes and increase their local concentration to accelerate pilus assembly [53]. The usher translocation pore forms an outer membrane β-barrel containing 24 TM (transmembrane) β-strands [52,53] (Figure 3C). The pore has an inner diameter of 45 Å×25 Å (1 Å=0.1 nm), a size compatible with the passage of folded subunits. Between strands 6 and 7 lays the plug domain, a βsandwich fold that, in an inactive usher, completely occludes the pore, preventing the passage of the subunits. Deletion of this domain completely abolishes the assembly of pili in vitro [53], thus the plug domain participates actively in pilus biogenesis. The C-terminal domain role is still not completely understood, but C-terminal truncation and deletion mutants of the P pilus usher PapC demonstrated that the C-terminus is fundamental for proper binding of chaperone–subunit complexes and pilus assembly [53,54]. Differences in protease susceptibility in the Type 1 pilus usher FimD show that following targeting to the usher N-terminus, the N-terminal domain of FimH (from the FimC–FimH complex) forms stable interactions with the FimD C-terminus, inducing a conformational change in FimD that may be fundamental in the activation step of pilus biogenesis [55– 57]. Such conformational change must include the movement of the plug either inwards within the translocation pore or outwards in the periplasm [52]. Binding of the chaperone-adhesin (PapDPapG) with the usher C-terminus was also demonstrated in vitro. Complementation studies on PapC C-terminal truncates have also shown that, in the absence of the C-terminal domain, the translocation pore is unable to become activated. These results suggest an important role of the C-terminal domain in the first step of pilus biogenesis, the activation of the usher [54]. The P pilus usher PapC assembles into ring-shaped homodimeric functional units in vivo. This was demonstrated by cryo-EM (where EM is electron microscopy) images of two-dimensional crystals where PapC was reconstituted in E. coli lipids and in complementation studies of PapC lossof-function mutants [54,58]. Dimer formation doesn’t seem to be necessary for fibre formation in vitro [53]. Cryo-EM reconstruction of FimD–FimH–FimG–FimF–FimC shows that only one of the ushers is activated and able to secrete subunits (Figure 3D) [52]. In fact, the requirement for a second usher in vivo is not completely understood. Remaut et al. [52] proposed a model where the two usher N-terminal domains alternate in the recruitment of new chaperone–subunit complexes. The structure of the C-terminal domain and of usher–chaperone–subunit complexes will provide answers to the mechanism of usher activation and assembly mechanism. TYPE IV SECRETION SYSTEM T4SSs are specialized assemblies utilized by both Grampositive and Gram-negative bacteria for one-step transport of Two-step versus one-step secretion systems Figure 3 479 The usher PapC structure (A) Domain organization of PapC. The translocation domain and the N-terminal domains (Ntd) are represented in dark blue, the plug domain in magenta, and the C-terminal domain (Ctd) in white. Residue boundaries are indicated. (B) Structure of the FimD N-terminal domain (dark blue) bound to the FimC–FimHp (red and light green respectively) complex (PDB: 1ZE3). FimHp is the pilin domain of FimH. (C) Structure of the translocation domain of the PapC usher (PDB: 2VQI). The domain is in ribbon representation colour-coded in dark blue for the barrel, yellow for the single helix present in the structure, orange for the trigger hairpin, and magenta for the plug domain. Upper panel: side views. Lower panels: top views. (D) Type 1 pili tip captured by cryo-EM. X-ray structures from FimH (light green), FimD (dark blue), FimG (orange), FimC (red) and FimF (yellow) were docked [52]. X-ray crystallography structures used were FimH (PDB: 1QUN), FimD (PDB: 2VQI), FimG (PDB: 3BFQ), FimC–FimF–FimDN (PDB: 3BWU) and the cryo-EM map of the FimD2:C:F:G:H type 1 pilus tip assembly intermediate is from emd_5009 (EM Data Bank). A three-dimensional interactive Figure of this structure is available at http://www.BiochemJ.org/bj/425/0475/bj4250475add.htm. protein, nucleic acid or nucleoprotein substrates across cell membranes [6,7,59,60]. They are evolutionarily related to bacterial conjugation systems. T4SSs are implicated in the transport of virulent proteins or DNAs in various plant, animal or human pathogens [61,62], in bacterial conjugation and in DNA uptake or release into the extracellular space [63,64]. T4SSs facilitate the exchange of genetic material, promoting genetic plasticity and adaptive response of bacteria to the environment. Conjugative transfer of antibiotic-resistance genes via T4SSs is a leading cause for emergence of multidrug resistance [65]. T4SSs are formed by at least 12 proteins, termed VirB1– VirB11 and VirD4 according to the prototypical T4SS from Agrobacterium tumefaciens, although some T4SSs are composed of just a subset of these proteins [60,66]. The VirB/VirD4 proteins can be grouped based on their function and location into energizing proteins, core translocation pore complex, pilusassociated proteins and other T4SS proteins. Energizing proteins include the ATPases, VirB4, VirB11 and VirD4. VirB6 to VirB10 proteins form the core translocation complex. VirB6, VirB8 and VirB10 are inner membrane proteins, whereas lipoproteins VirB7 and VirB9 were thought to form the outer membrane pore. Most T4SSs form an extracellular pilus formed by the major VirB2 and minor VirB5 proteins. The other T4SS proteins include the putative lytic transglycosylase VirB1 and membrane protein VirB3, the location and function of which are still either ambiguous (location) or unknown (function). Structure and role of the various subunits The VirB4, VirB11 and VirD4 ATPases The three ATPases VirD4, VirB4 and VirB11 power T4SSs. They are located in the cytoplasm, sometimes inserted in the inner membrane, and form a network of interactions among themselves and the rest of the assembly [67]. c The Authors Journal compilation c 2010 Biochemical Society 480 Figure 4 A.T. Rêgo, V. Chandran and G. Waksman Atomic structures of known T4SS components Top panels, the crystal structures in cartoon representation for the E. coli VirB5 homologue TraC (PDB: 1R8I), B. suis VirB8 (PDB: 2BHM), H. pylori VirB10 homologue ComB10 (PDB: 2BHV) and the NMR structure of E. coli VirB9/TraO C-terminal domain in complex with VirB7/TraN (PDB: 2OFQ). The lower panels show in cartoon representation, the oligomeric and monomeric crystal structures of the H. pylori VirB11 homologue HP0525 (PDB: 1NLZ) and the E. coli VirD4 homologue TrwB from plasmid R388 (PDB: 1E9S). CTD, C-terminal domain; NTD, N-terminal domain. VirD4 VirD4 proteins are the cytoplasmic gatekeepers that recruit T4SS substrates to the T4SS channel. VirD4s are inner membrane proteins with an N-terminal TM domain and a large cytoplasmic domain [68]. TrwB, encoded by the IncW conjugative plasmid R388, has been the most characterized VirD4 homologue. The TM domain of TrwB plays a role in stability, substrate selectivity and oligomerization of the protein [69,70]. The crystal structure of the cytoplasmic soluble domain of TrwB [71] revealed a large 110 Å × 90 Å globular hexameric assembly with a 20 Å channel (Figure 4). This channel narrows down to an ∼ 8 Å constriction at the cytoplasmic end. Each subunit is shaped like an orange slice and made up of two distinct domains, an NBD (nucleotidebinding domain) and an AAD (all-α-helical domain). The NBD domain is a mixed α/β RecA-like domain with a central highly twisted nine-stranded β-sheet flanked by α-helices on both sides and orientated towards the inner membrane. Both the Walker A and B motifs for NTP-binding are essential for secretion [72]. A DNA-dependent ATPase activity has been demonstrated for TrwB [73]. No helicase activity has been observed so far for VirD4s. The AAD domain, consisting of seven α-helices, is orientated towards the cytoplasm. A DNA-binding role for this domain has been suggested, based on structural homology. VirB11 VirB11 belongs to a large family of traffic ATPases involved in various secretion systems such as type II, III, IV and VI [66,74,75]. These NTPases are important for their ATP-bindingdependent substrate selection and translocation, and for pilus production [76]. VirB11-like proteins exhibit distinct substrate c The Authors Journal compilation c 2010 Biochemical Society specificity and their NTPase activity is enhanced by the presence of detergent/phospholipids in vitro [77,78]. VirB11-like proteins have indeed been shown to be peripheral membrane proteins associated with the inner membrane [79]. Crystal structures have been solved for the Helicobacter pylori VirB11 homologue HP0525 in its apo and substrate-bound forms, and for the Brucella suis VirB11 protein [80–82] (Figure 4). Each HP0525 monomer is made up of two domains: an N-terminal domain with a novel fold and a C-terminal domain with the typical RecA fold. The two domains form hexameric rings mounted on each other shaped like a six-clawed grapple. The N-terminal domain consists of a central six-stranded β-sheet packed against two α-helices on one side, whereas the C-terminal domain is made up of a seven-stranded β-sheet sandwiched between three and four α-helices on either side. The interface between the two domains forms the NTPbinding site. VirB11 from B. suis shows a very similar double hexameric ring structure with the individual domains in each monomer structurally similar to those of HP0525 [82]. However, a large domain swap is noticed that leaves the overall hexamer intact but alters both the substrate-binding site and inter-subunit interfaces. The apo and substrate-bound structures of HP0525 have given valuable structural insight into the function of VirB11like ATPases [80,81,83]. The apo-form of HP0525 shows an asymmetric hexamer due to rigid body movements in the NTD ring of the assembly. The conformational flexibility has been suggested to help further interactions with the rest of the T4SS assembly. ATP binding takes place initially at three catalytic sites, giving rise to a more rigid conformation to the entire assembly. ATP hydrolysis at these sites followed by ATP binding to the remaining three sites locks the entire assembly into a perfectly Two-step versus one-step secretion systems symmetrical hexamer. Further hydrolysis and release of ADP from all sites returns the molecule to its initial asymmetric apo-form. VirB4 VirB4-like proteins are the largest ATPases in T4SSs and are evolutionarily conserved in both Gram-negative and Grampositive bacteria [6,84]. Bioinformatic studies suggest four conserved motifs, including the Walker A and B motifs and a conserved domain for the VirB4 family of proteins [85]. The NTPbinding motifs are essential for the functionality of T4SSs [86]. However, unambiguous demonstration of the ATPase activity for VirB4 homologues has been difficult, with two positive reports for A. tumefaciens VirB4 and more recently TrwK from plasmid R388 [87,88]. No structural detail is available for VirB4-like proteins and there is ambiguity regarding the exact cellular location and oligomerization state. Topological studies on A. tumefaciens VirB4 map it as a polytopic cytoplasmic membrane protein with probably two periplasmic and three cytoplasmic domains [89]. TrwK, the VirB4 homologue from plasmid R388, however, is a soluble protein [88]. A hexameric model of the C-terminal domain of VirB4 has been proposed based on its relationship to the cytoplasmic domain of VirD4 homologue TrwB by divergent evolution [90]. This initial model suggested a cytoplasmic location for VirB4, although subsequent work places the C-terminal domain predominantly in the periplasm [91]. Core channel components VirB6 VirB6 is a highly hydrophobic inner membrane channel subunit essential for T4SS. No structural information is available to date for this T4SS component. Topological studies on both A. tumefaciens VirB6 and H. pylori ComB6 have mapped an N-terminal periplasmic domain, five TM segments and a cytoplasmic C-terminal domain [92,93]. A large periplasmic loop (P2 loop) in the centre of the protein between TM2 and TM3 presumably contributes to the inner walls of the transport channel. The C-terminal TM segments orchestrate substrate transfer to the other inner membrane component VirB8, whereas both terminal regions co-ordinate with the outer membrane and pilin subunit VirB9 and VirB2. The physiological oligomeric state of VirB6 itself is not known. VirB8 VirB8 is a bitopic inner membrane protein with a short cytoplasmic tail, a single TM helix and a large periplasmic domain [68]. VirB8 is thought to serve as the nucleation centre for the assembly of T4SSs [60,94]. Immunofluorescence and immunoelectron microscopic methods showed unique clustering of VirB8 gold particles independent of other VirB proteins and also the influence of VirB8 on VirB9–VirB10 localization [94]. VirB8 participates in a rich network of protein–protein interactions in the T4SS with VirB1, VirB4, VirB5, VirB9, VirB10 and VirB11, and is in direct contact with the substrate during transport [94–100]. High-resolution structural information is available for the periplasmic domains of two homologues, A. tumefaciens and B. suis VirB8 (Figure 4) [101,102]. Both crystal structures reveal a dimer of VirB8 with an extended four-stranded antiparallel β-sheet juxtaposed by five α-helices. The periplasmic domains have a fold similar to nuclear transport factor NTF2. The crystal 481 structures reveal a deep groove lined by highly conserved residues, a structural feature absent in NTF2 and other similar fold proteins and is probably a hot spot for protein–protein interactions. Mutational analysis of VirB8 identified a β-sheet region adjacent to this groove to be important for interactions with VirB4 and VirB10 [103]. The physiological oligomeric state of this protein in the T4SS assembly is still unknown. VirB10 VirB10 is also a bitopic inner membrane protein with a similar topology as VirB8 with a short N-terminal cytoplasmic tail, a single predicted TM helix and a large periplasmic domain consisting of a proline-rich region and a β-barrel domain [68,104]. VirB10, although inserted in the inner membrane, has also been observed associated with the outer membrane [94]. Interactions of VirB10 with VirB9 [100] and the energizing proteins VirB4, VirB11 and VirD4 [67,105–108] substantiated by mutational analysis have established VirB10 as the structural scaffold of T4SSs bridging the two membranes together [104]. A high-resolution crystal structure is available for part of the periplasmic domain of H. pylori ComB10 protein [102] (Figure 4). This C-terminal periplasmic domain consists of an atypical β-barrel flanked by an α-helix on one side and an α-helical antennae-like projection at the top. The altered β-barrel is splayed open on one side. A head to tail dimer is observed in the crystal structure, which is probably a crystal-packing artefact. Mutational studies have highlighted the role of the side helix and α-helical antennae in uncoupling pilus biogenesis and substrate transfer functions of T4SSs [104]. See Note added in proof. VirB10 shares features with the inner membrane energy sensor protein TonB: both are bitopic membrane proteins with a prolinerich region and conserved hydrophobic or coiled-coil regions important for protein–protein interaction and function. Similar to TonB, VirB10 probably couples inner membrane energy status to the outer membrane gating required for transport. Indeed, VirB10 responds to the cellular ATP levels with different protease susceptibility, suggesting different structural conformations. The structural transition of VirB10 in response to ATP levels also affects the VirB7–VirB9 heterodimer and VirB7–VirB9–VirB10 complex formation [109]. VirB7 and VirB9 VirB7 is a monotopic lipoprotein [110]. The modified N-terminus is anchored in the outer membrane and the C-terminus is periplasmic. In A. tumefaciens, VirB7 stabilizes VirB9 by forming a disulfide link between the two proteins [111]. VirB9 is mainly a periplasmic protein that has been suggested to form the outer membrane pore, although no TM segments have been predicted. Mutational studies on A. tumefaciens VirB9 has mapped the N-terminal domain to be important for substrate selection and the C-terminal domain for stabilizing the VirB10–VirB9–VirB2 proteins that form the distal end of the channel and are essential for secretion [112]. The NMR structure has been solved for the interacting domains, for example the C-terminal domain of the VirB9 homologue encoded by the E. coli plasmid pKM101, TraO (TraO-CT), bound to the VirB7 homologue TraN encoded by the same plasmid (Figure 4) [113]. The structure reveals a β-sandwich fold for TraO-CT, around which TraN wraps. Two edge strands in TraO that diverge away from the β-sandwich fold form an appendage which has been shown to be surface exposed in A. tumefaciens, suggesting a role in outer membrane pore formation. See Note added in proof. c The Authors Journal compilation c 2010 Biochemical Society 482 A.T. Rêgo, V. Chandran and G. Waksman Pilus components The function of the type IV secretion pilus (not to be mistaken for the type IV pilus, the assembly of which is a T2SSrelated process) is still unclear. It might be used as conduit for substrates [13,114] or adhesion [115,116]. The T pilus of A. tumefaciens consists of a processed form of VirB2, the major component of pilin, and VirB5, the minor component [117,118]. Pilus biogenesis requires other T4SS components and several uncoupling mutations have been mapped that allow secretion but not pilus assembly [76,104,112]. VirB2 is highly processed: the N-terminal leader signal peptide that targets the pilus subunit across the inner membrane is cleaved by specific peptidases and further processing might occur, sometimes including cyclization [119–122]. No structural information for VirB2-like proteins alone is available. However, a crystal structure is available for the VirB5 homologue in pKM101, TraC (Figure 4C) [123]. The structure reveals a three-helix bundle capped by a globular appendage. Structure-based mutagenesis studies suggested a role of VirB5 in adhesion and host recognition [123]. Recently, the F pili structure has been determined using cryo-EM and single-particle reconstruction [124]. Two pilin subunits pack with two different symmetries that co-exist; a C4 symmetry forming stacked rings axially separated by ∼ 12.8 Å and a one-start helical symmetry with an axial rise of 3.5 Å per subunit and a pitch of ∼ 12.2 Å. The central lumen seems large enough for transferring single-stranded DNA but not folded proteins. Other T4SS-associated proteins VirB1 VirB1, although non-essential for secretion, is essential for pilus biogenesis [125–127]. Deletions of the virB1 gene lead to attenuated virulence with respect to T4SS functions [99]. An exception in this case is that of H. pylori, where the VirB1 homologue, HP0523, is essential to bacterial virulence [128]. VirB1 is a periplasmic protein with a conserved lysozyme-like transglycosylase domain [129]. The catalytic activity of the transglycosylase domain to lyse the peptidoglycan cell layer probably helps to accommodate the large T4SS assembly in the periplasmic space. VirB1 is processed [130] to release a C-terminal third of the protein VirB1* which is required for pilus biogenesis and is secreted to the extracellular space [127]. Two hybrid experiments and later biochemical experiments have shown interaction of VirB1 with other T4SS components VirB9, VirB8 and VirB11 suggesting more (as yet undefined) roles for VirB1 in T4SS assembly and function [99]. VirB3 VirB3 is one of the most ill understood proteins in the T4SS assembly both in terms of localization and function. It is a membrane protein with TM segments predicted near the N-terminus. Localization of this protein is very uncertain, with reports suggesting either inner or outer membrane localization [131,132]. Chimaeric VirB3/VirB4 proteins previously identified in certain T4SS assemblies such as Campylobacter jejuni [133] suggest an inner membrane localization to be more probable due to association with VirB4, which is associated with the inner membrane. However, no functional role has been identified so far for this protein. Assembly of T4SSs The biogenesis of the T4SS assembly has been proposed to start with the formation of a core complex (VirB6–VirB10) (Figure 5), c The Authors Journal compilation c 2010 Biochemical Society followed by recruitment of the ATPases (VirD4, VirB4 and VirB11) and/or pilus biogenesis (VirB2 and VirB5). Secretion and pilus biogenesis might require two different complexes [6,7], and thus, after assembly of the core complex, there might be a bifurcation in the assembly process leading to formation of two different complexes, one used for secretion, the other used for pilus biogenesis. Indeed, mutations in some VirB proteins decouple secretion from pilus biogenesis [76,92,104,112]. Also, VirD4 is only used in secretion, whereas VirB11 is only used in pilus biogenesis [127,134]. However, at this stage, the exact composition of these complexes is not known. A recent breakthrough in the field has been the 15 Å cryoEM structure of the core translocation channel formed by the VirB7, VirB9 and VirB10 homologues of the E. coli pKM101 plasmid (TraN–TraO–TraF) (Figure 5) [135]. This provided the first snapshot of a T4SS assembly. The core complex is a 1.05 MDa structure with 14-fold symmetry and traverses across both inner and outer membranes. The nucleation factor VirB8/TraE, although co-expressed with the other proteins, is not required for forming the core complex. The lipidation of lipoprotein VirB7/TraN is also not essential for core complex formation, but required for targeting the complex to the outer membrane. The structure shows multilayered rings consisting of 14 copies each of TraN/VirB7, TraO/VirB9 and TraF/VirB10, with a height and diameter of 185 Å. Two distinct layers are seen, termed the I (inner)- and O (outer)-layers with respect to location in the membrane (Figure 5). The two layers are partly or fully double walled. The O-layer consists of a cap and a main body. The cap, which probably traverses the outer membrane, encloses a 20 Å pore that narrows to 10 Å at the junction with the main body. The main body has a larger internal chamber that is 110 Å wide narrowing down to 55 Å at the base. The I-layer, that resembles a cup, has a partial double wall that merges down at the base and encloses a chamber similar to the O-layer. A total of 14 finger-like densities extend below the I-layer. Using various biochemical studies, the C-terminal domains of both VirB9 and VirB10 have been located in the O-layer and the N-terminal domains of both proteins in the I-layer. The finger-like protrusions probably are the TM segments of TraF inserted in the inner membrane. T4SS mechanism One of the most valuable contributions to the T4SS field has been the TrIP (transfer DNA immunoprecipitation) study on the A. tumefaciens T4SS, delineating the substrate transfer route [126]. In A. tumefaciens, the substrate is the T-DNA (transferred DNA). It successively contacts VirD4, VirB11, VirB6, VirB8, VirB2 and VirB9 during transfer. Other T4SS components, VirB3, VirB4, VirB5, VirB7 and VirB10, assist the transfer indirectly, modulating/regulating differentially each of the transfer steps. The coupling protein VirD4 contacts the substrate first, independently of ATP hydrolysis [67]. The substrate is then passed on to VirB11, independently of any energy requirements. The lipoprotein VirB7 is important for this initial step of transfer, highlighting its importance for mediating the formation of the stable core assembly. The substrate then comes in to contact with the inner membrane proteins VirB6 and VirB8. This early transfer depends on the energetic components VirD4, VirB4 and VirB11, and ATP hydrolysis is required at this stage. This seems to suggest that VirB6 and VirB8 function together and probably line the inner surface of the VirB7–VirB9–VirB10 core complex to contact the substrate directly. The substrate is then transferred to the outermembrane-associated proteins VirB9 and the major pilin subunit VirB2. VirB3, VirB5 and VirB10 are important at this stage of transfer. The importance of VirB10 is evident from the fact that it Two-step versus one-step secretion systems Figure 5 483 A model for Type IV secretion systems A model for the assembly of T4SS components VirB1–VirB11/VirD4 is shown. A cut-away view of the cryo-EM structure of the core translocation assembly formed by VirB7/VirB9/VirB10 homologues in E. coli pKM101 is shown in grey crossing both the inner and outer membranes. A surface representation of the atomic structures of T4SS components (VirB11 homologue, PDB entry 1NLZ; VirD4 homologue, PDB entry 1E9S; VirB5 homologue, 1R8I; and VirB8, PDB entry 2BHM) that are available are shown. The T4SS components for which no known structural information is available are shown in distinct shapes and colours. The three ATPases powering the T4SS on the cytoplasmic side of the membrane are shown. Inner membrane components VirB6 and VirB8 are shown lining the inner surface of the core complex. Pilus-associated proteins VirB2 and VirB5 and other proteins VirB1 and VirB3 are shown. All components are labelled according to their A. tumefaciens VirB/VirD4 nomenclature. bridges the two membrane components together (Figure 5) [104] and plays the role of an energy sensor for bringing about the required conformational changes important for substrate transfer [109]. VirB5 is a minor pilin subunit probably assisting the passage of substrate via the pilus. Thus the entire pathway for substrate translocation has been defined and, except for VirB1, all proteins play an important role for secretion. CONTRASTING THE TWO SYSTEMS Simplicity compared with complexity in the translocation of substrates Secretion systems range from relatively simple structures, such as the CU secretion system composed of two subunits (the chaperone and the usher) specialized in assembling and driving the secretion of pilus subunits, to more complex machineries such as T4SS, which is composed of at least 12 subunits able to mediate secretion of a wide variety of substrates. T4SS proteins span both the inner and outer membranes, forming MDa multicomplex machineries, whereas the CU system proteins only form complexes with the chaperone when they are recruited, and then with the usher and each other to form the pilus structure. The translocation pore in the CU system is composed simply of a ∼ 180 kDa outer membrane dimeric usher [52] that works as an assembly platform. The pore diameter of one of the PapC monomers (45 Å × 25 Å) is large enough to allow folded subunits translocation (ranging from 27 Å × 22 Å to 32 Å × 23 Å in diameter for PapA and PapK respectively) upon movement of the plug domain. How the plug is let go is not known, but simple mechanical processes might operate such a release, probably mediated by the interaction of the first chaperone–subunit to be presented to the usher (the chaperone–adhesin complex) with the C-terminal domain of the usher. In contrast, the translocation pore complex in T4SSs, in its minimal form, involves at least three proteins (VirB7, VirB9 and VirB10) with 14 copies of each assembling into a 1.05 MDa channel complex [135]. At least on one side (the O-layer), the channel is closed and, thus, it must open to let substrates through. This necessitates complex multi-proteins sensing and mechanical devices to operate the conformational changes required to drive secretion of substrates, probably mediated by VirB10, but also other VirB proteins not contacting the substrate. ATP dependency/independency Protein secretion across the outer membrane is dependent on an energy source. However, the most commonly used energy store in living organisms, ATP, is not present in the bacterial c The Authors Journal compilation c 2010 Biochemical Society 484 A.T. Rêgo, V. Chandran and G. Waksman periplasm. In the T4SS several cytoplasmic components are assembled in a large cytoplasmic/inner membrane ATPase complex (VirD4, VirB4 and VirB11) that utilizes ATP to operate the conformational changes required to drive secretion across both the inner and outer membranes. In the CU system no cytoplasmic or inner membrane components are encoded and no ATP is used. In fact, no external source of energy is required for DSE and consecutive pilus formation [44]. Instead, the chaperone stabilizes a high-energy conformation of the bound subunit, which upon DSE is released, resulting in an exceptionally stable and closed subunit conformation. The free energy released during the process is believed to be sufficient to drive pilus formation [43]. It should not be inferred from the comparison above that, in all two-step secretion systems, transport through the outer membrane is ATP-independent. For example, in type II secretion, although substrates are dependent on Sec or Tat (and thus ATP) for transport through the inner membrane, transport through the outer membrane also requires ATP. Although this is not completely understood, it has been proposed that passage through the outer membrane pore (termed the ‘secretin’) is powered by pilus growth and retraction, a process dependent on inner membrane ATPases and consumption of ATP. Thus, in T2SS, an active ATP-dependent mechanism utilizing the pilus as a piston might mechanically push substrates through the outer membrane secretin [4]. Conversely, not all one-step secretion systems are dependent on ATP. For example, although one-step transport by some T1SS are powered by ATP, other T1SSs might use the inner membrane proton gradient as a source of energy [136,137]. The present review does not have the space to expand the description of these systems, but it must be realized that, although common features are clearly shared among secretion systems, each also exhibit unique characteristics [138]. Chaperone presence and dependency Molecular chaperones have been characterized in all living cells as accessory proteins in various cellular functions. These include de novo protein folding, stabilization of proteins under stress conditions and maintenance of a loosely folded state for translocation across membranes. Cytoplasmic chaperones are involved in different types of protein secretion. They have been best characterized in T3SSs where they are thought to maintain the stability and secretion competence of effector proteins, but have also been found in the T4SS. For example, the VirE1 protein of A. tumefaciens is necessary for the translocation of the effector protein VirE2 into plant cells [139–143]. Other ‘type IV adaptor proteins’ required for translocation of a subset of substrates, such as IcmS, IcmW and LvgA proteins of the L. pneumophila Icm/Dot T4SS [144–146] may have a similar function. CagF is another candidate for secretion chaperone-like molecule (present in both soluble and membrane-associated milieu) of the CagA protein [147]. In the periplasm, chaperone function is distinct from the cytoplasmic chaperones and independent of any known source of cellular energy. PapD-like periplasmic chaperones are small monomeric proteins that transiently contribute steric information to pilus subunits to facilitate their folding, maintain them in a high-energy state, and prime them for future assembly. In the CU secretion system, the role and mechanism of the chaperone is very well understood and a single chaperone seems to be enough to drive all of the different subunits to the assembly platform. On the other hand, in T4SSs and other systems, chaperones are still not very well characterized and their function is still poorly understood. c The Authors Journal compilation c 2010 Biochemical Society Type IV secretion pilus compared with CU pilus T4SS pili are varied in shape and size. The conjugation pili in T4SS systems are classified based on morphology into two classes: the long flexible F-type pili and the short rigid P-type pili [148]. The F-type T4SS pili measure 2–20 μm in length and 8–9 nm in diameter with a 2 nm central lumen [149,150]. The P-type pili are shorter than 1 μm in length and 8–12 nm in diameter [149,150]. The overall structure of a F-type pilus has been determined (see above) and the subunits appear to stack in a superhelical arrangement. On the other hand, the H. pylori T4SS system encoded by the Cag pathogenicity island produces distinctive, large sheathed pili, ∼ 100–200 nm long and ∼ 70 nm wide with a ∼ 45 nm central needle made up of a VirB10 homologue (HP0527/CagY) [115,128,151]. It is believed that T4SS pili essentially play an adhesion role. For example, CagL, possibly the VirB5 homologue of the Cag-encoded T4SS, is targeted to the pilus surface and appears to play a role in host recognition [115]. However, the role of T4SS pili as adhesive devices has not been as clearly demonstrated as in the CU pili systems, notably the P and Type I CU pili systems. Thus it remains unclear whether T4SS pili are used for adhesion or as conduit for substrate secretion or both. CU systems also encode a variety of pilus types ranging from the rigid rod-like pili, the detailed description of which is provided above (P and Type 1 CU pili), but also very thin fimbriae or filaments forming mingled masses at the surface of the bacteria that harbour them [40,42,152,153]. In P and Type 1 pili, only one protein is responsible for adhesion and host recognition (PapG and FimH respectively), and this protein locates at the pilus tip due to the unique properties of the chaperone–adhesin complex to activate the usher. However, in some other CU pili systems where only one or two pilus subunits have been identified, these are likely to combine the functions of structural and adhesive subunits, i.e. they assemble to form a pilus or fimbria but also act as adhesins. In that case, the adhesive property of the pilus would no longer be confined to the tip but thought to be spread out over the entire length of the pilus/fimbriae [154]. PROSPECTS We have used two different secretion systems, the CU pilus system and the T4SS, to contrast two classes of secretion systems, those using two distinct machineries to release substrates first through the inner membrane and next through the outer membrane (CU pili), and those that elaborate a complex double membranespanning machinery to cross both membranes (T4SSs) in a single step. In the two-step systems, the Sec translocon is used to cross the inner membrane. No details on the Sec machinery is provided in the present review as it has been excellently documented elsewhere [28,29]. Subsequent transport through the outer membrane involves two specialized proteins, a chaperone and an usher. Both proteins are used to drive a process of assembly and secretion, which is made energetically favourable by the exquisite modulation of protein folding by the chaperone. By maintaining the subunits in a semi-unfolded conformation, the chaperone stores up energy which, when released upon DSE, drives the pilus biogenesis process forward. Processes involving folding energy to drive secretion are also at play in Type V secretion, a secretion process through the outer membrane, which requires a single transporter polypeptide forming a β-barrel in the outer membrane. Type V secretion substrates, often (but not always) are part of the same polypeptide that forms the β-barrel, are driven through the barrel in an unfolded state, and folding Two-step versus one-step secretion systems post-transport is thought to drive the extrusion of the unfolded peptide. Because T4SSs are double membrane spanning, they have access to the store of ATP in the cytoplasm and thus the T4SS ATPases power secretion probably through complex, undiscovered macromolecular relays regulating pore opening and closure at both the inner and outer membranes. It will be one of the challenges in the next few years to elucidate the molecular basis of such relays. In this respect, the challenge extends to all secretion systems, whether in bacteria or eukaryotes. It is likely that within one class of secretion systems, common features will be discovered, as well as distinct ones, but expanding knowledge in this field is bound to provide inspiration for designing more efficacious secretion systems inhibitors, which, at least in the case of bacterial pathogens, are urgently needed. Note added in proof (received 3 December 2009) The structure of the outer membrane complex of the T4SS encoded by the pKM101 plasmid has just been published [156]. This complex is made of the assembly of 14 copies of each of the TraN/VirB7 protein, the C-terminal domain of the TraO/VirB9 protein and the C-terminal domain of the TraF/VirB10 protein. 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