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A comprehensive guide to pilus biogenesis in Gram-negative

Manuela K. Hospenthal, Tiago R. D. Costa and Gabriel Waksman Abstract | Pili are crucial virulence factors for many Gram-negative pathogens. These surface structures provide bacteria with a link to their external environments by enabling them to interact with, and attach to, host cells, other surfaces or each other, or by providing a conduit for . Recent high-resolution structures of pilus filaments and the machineries that produce them, namely chaperone–usher pili, type IV pili, conjugative type IV secretion pili and type V pili, are beginning to explain some of the intriguing biological properties that pili exhibit, such as the ability of chaperone–usher pili and type IV pili to stretch in response to external forces. By contrast, conjugative pili provide a conduit for the exchange of genetic information, and recent high-resolution structures have revealed an integral association between the subunit and a phospholipid molecule, which may facilitate DNA transport. In addition, progress in the area of cryo-electron tomography has provided a glimpse of the overall architecture of the type IV pilus machinery. In this Review, we examine recent advances in our structural understanding of various Gram-negative pilus systems and discuss their functional implications.

Pili Gram-negative bacteria assemble various non-flagellar Chaperone–usher pili Long non-flagellar appendages surface termed ‘pili’ or ‘fimbriae’. These struc- Chaperone–usher pili are ubiquitously expressed on the at the surface, also referred tures are long, thin proteinaceous polymers that carry out surface of many Gram-negative bacterial pathogens8. to as fimbriae, that are present diverse functions and are crucial virulence factors during They are important virulence factors that facilitate in a wide range of Gram- 1 negative and Gram-positive infection . Pilus assembly is coordinated and catalysed by host–pathogen interactions that are crucial for the estab- bacteria and in , and elaborate multiprotein complexes that either span both lishment and persistence of an infection, and for other are involved in bacterial the bacterial inner membrane and outer membrane or key processes such as formation1,5. The role of attachment, and are localized in the outer membrane only2. Moreover, chaperone–usher pili in the pathogenesis of urinary tract . the structures and functions of the resulting pili vary infections (UTIs) caused by uropathogenic Escherichia and reflect bacterial adaptations to unique environmental coli (UPEC) has been particularly well documented9,10. conditions. A common function of pili is to mediate con- UPEC uses two types of chaperone–usher pili, type 1 and tact between bacteria and host tissues during infection, P pili5, which will be described here as models that high- between bacteria and other surfaces, or between neigh- light the structure and assembly of surface organelles bouring bacteria3. For example, conjugative pili facilitate manufactured by the chaperone–usher pathway. During contact between a donor and an acceptor bacterium, infection with UPEC, type 1 pili mediate interactions thus enabling the cell‑to‑cell transport of DNA, whereas with the bladder, whereas P pili target the kidney. Both contact that is mediated through other types of pili may types contribute to the ability of UPEC to ascend from lead to infection, , or micro­colony or the bladder to the upper urinary tract during infection11. biofilm formation1,4–7. This Review focuses on the diverse Institute of Structural and structures and functions of assembly machineries that are The biogenesis of chaperone–usher pili. Type 1 and P pili Molecular Biology, University involved in the biogenesis of five known types of pili in are assembled from distinct pilus subunits, or ‘’, t h a t College London and Birkbeck, Gram-negative bacteria. These are the chaperone–usher are encoded in the fim and pap operons, respectively12. University of London, Malet type IV pili Street, London WC1E 7HX, pili, the (T4P), the conjugative type IV secre- Type 1 and P pili are organized into two subassemblies; UK. tion pili, curli fibres (BOX 1) and the recently described a short, thin tip fibrillum mounted on a helical rod that 5 (FIG. 1a) Correspondence to G.W. type V pili. In particular, we focus on recent structural is 1–2 μm in length . Not all chaperone–usher g.waksman@ progress that has enhanced our understanding of the pili have the same architecture; some lack the tip fibril- mail.cryst.bbk.ac.uk molecular mechanisms that lead to pilus assembly and lum, whereas others lack a rod structure13. At their distal doi:10.1038/nrmicro.2017.40 how the biophysical properties of the assembled pili end relative to the outer membrane, type 1 and P pili Published online 12 May 2017 ­contribute to their functions. contain an adhesin protein (FimH for type 1 pili and

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PapG for P pili; FIG. 1a). These adhesins are composed 5–10 PapE subunits and one PapK subunit for P pili. The of two domains, an amino‑terminal ­lectin domain that majority of the pilus length is formed by the rod, which enables bacteria to interact with specific host cell recep- comprises ~1,000 copies of a single ­subunit, either FimA tors and a ‘pilin’ domain that engages with the next pilin for type 1 pili or PapA for P pili5 (FIG. 1a). sub­unit14,15. The assembly of a complete tip fibrillum Individual chaperone–usher pilins are transported requires the sequential addition of either a single subunit across the inner membrane into the by the of FimG and FimF for type 1 pili, or one PapF subunit, general secretory pathway (Sec pathway), which uses the SecYEG translocon16. Following transport across the inner membrane, pilins are folded and stabilized by dedi- Box 1 | The mechanism of curli fibre formation cated periplasmic chaperones, either FimC for type 1 pili (FIG. 1a) Curli are extracellular functional amyloid fibres that are assembled by enteric bacteria. or PapD for P pili . Unbound subunits are unsta- They have roles in pathogenicity and the formation of . The complex extracellular ble because they form incomplete immunoglobulin-­like matrix that is formed in a biofilm protects the bacterial community from conditions of folds that are comprised of six β‑strands; these lack the external stress and promotes colonization and persistence. Curli fibres are assembled by seventh carboxy‑terminal strand that is usually found a dedicated secretion pathway that begins with the transport of unfolded CsgA subunits in immunoglobulin‑like folds. The lack of this strand to the periplasm through the SecYEG translocon124 (see the figure). In the periplasm, results in a hydrophobic groove that runs along the CsgA subunits have three possible fates. They can progress to the CsgG channel en route length of the pilin14,17. This groove contains five hydro- to the bacterial surface, undergo proteolytic degradation or remain in the periplasm, phobic pockets (P1–P5 pockets), which can be com- where the polymerization of CsgA that would lead to the formation of toxic fibres is plemented by hydrophobic residues (P1–P5 residues) prevented by CsgC binding to CsgA7. At the periplasmic side of the outer membrane, CsgA subunits first interact with the nonameric CsgE capping adaptor, before interacting present on a donor β‑strand that originates from either with the CsgG diffusion channel (a 36‑stranded nonameric β‑barrel pore) (Protein Data the chaperone before pilus assembly or from another Bank (PDB) entry 4UV3)125,126. When bound to CsgG, CsgE acts as a plug, but also as a pilin subunit after pilus assembly. The process by which secretion adaptor that assists CsgA subunits during their periplasmic transit127. When a periplasmic chaperone complements this groove by CsgA is enclosed in the CsgG–CsgE cavity, an entropy gradient inside the channel donating one of its own β‑strands is termed donor- promotes the diffusion of unfolded CsgA from the confined cage to the bacterial outer strand complementation­ (DSC)14,17–19. In the chaperone–­ surface7. At the surface, CsgB controls the nucleation and polymerization of CsgA subunit complex, the donor strand of the chaperone molecules into curli fibres in a CsgF-dependent manner128,129. It is unclear whether curli occupies the P1–P4 pockets of the groove of the sub- subunits are incorporated at the proximal or distal end of the curli fibre. unit, leaving the P5 pocket free. Once engaged in this complex, the chaperone then shuttles the subunit to an CsgA outer membrane-embedded nanomachine known as the usher, which is FimD for type 1 pili and PapC for P pili (FIG. 1a). At the usher, pilin subunits polymerize into a pilus that gets secreted through the usher pore. n At the usher pore, polymerization occurs through a process called donor-strand exchange (DSE)20–22. During DSE, the donor strand of the chaperone is replaced CsgB by the N‑terminal extension of the next subunit dur- ing assembly. The N‑terminal extensions of subunits OM CsgF are N‑terminal sequences that are 10–20 residues in length and are found in each sub­unit except for the tip adhesin. After DSE, the N‑terminal extension provides the donor strand for the previously assembled subunit, CsgG which engages the P1–P5 pockets in the groove of the PG channel previously assembled subunit through its corresponding­ Toxic CsgA fibres P1–P5 residues20,23. CsgE CsgC Proteolysis Pilus assembly at the usher. The process of pilus subunit polymerization is the same for type 1 pili and P pili, and occurs at the outer membrane usher. The usher itself is composed of a 24‑stranded β‑barrel pore, a periplasmic CsgA Periplasm N‑terminal domain (NTD), two periplasmic C‑terminal domains (CTD1 and CTD2) and a plug domain24,25 (FIG. 1b). The first step of pilus biogenesis is the recruit- IM CsgA SecYEG ment of a chaperone–adhesin complex to the NTD of Translocon 26–28 FIG. 1b Bacterial cytosol the usher ( , step 1). This step was captured crystallographically by a ternary complex of the usher

NTD (FimDNTD) bound to a chaperone–pilin com-

plex, FimDNTD–FimC–FimHP (FimHP refers to the IM, inner membrane; OM, outer membrane; PG, . Figure is adapted with permission FimH pilin domain), which showed that the inter­ Nature Reviews | Microbiology from REF.7, Elsevier. action between the chaperone–adhesin complex and

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the NTD of the usher occurs predominantly through it is clear that the two domains of FimH (the lectin and the chaperone29. However, the affinity of the chaper- pilin domains) undergo a major conformational change one–adhesin complex for the NTD is higher than for relative to each other. In the pre-transport conforma- any other chaperone–subunit complex, which suggests tion, the pilin domain of FimH is aligned with the FimH that the adhesin may also have a role in usher–NTD lectin domain, whereas it moves 37.5° with respect to its binding27,29. Next, the chaperone–adhesin complex is original position once extruded from the usher pore34. transferred to the CTDs of the usher (FIG. 1b, step 2). This conformational change is thought to prevent the This state was captured in a crystal structure of full- tip fibrillum from slipping back into the usher; however, Biofilm 30 A community of bacterial cells length FimD bound to the FimC–FimH complex . this hypothesis remains to be tested. that form a dense surface- At this stage, the usher plug domain (which, in the The host receptors that are targeted by the type 1 associated matrix of proteins, unbound form of the usher, is located inside the usher pilus adhesin FimH include d‑mannosylated recep- nucleic acids and pore) was displaced by FimH and moved into a position tors, such as the uroplakins of the bladder, whereas polysaccharides that provides proximal to the NTD of the usher, whereas the lectin the P pilus adhesin PapG targets galabiose-containing a strong fitness advantage, such as an enhanced tolerance domain of FimH was inserted inside the usher pore. In glyco­sphingolipids, which are primarily located on the 37–39 to and a reduced the next step of pilus biogenesis, another chaperone– kidney epithelium . Therefore, the receptor specificity susceptibility to host immune subunit complex is recruited to the NTD of the usher of the type 1 and P pilus lectin domains may contribute responses and other physical (FIG. 1b, step 3). When this step is modelled onto the to the observed tropism of UPEC for the bladder or the and chemical stresses. FimD–FimC–FimH structure, it becomes clear that the kidneys during the course of an infection40. In addition, Type IV pili N-terminal extension of the incoming subunit is opti- FimH can transition from a low-affinity binding state to (T4P). Widespread surface mally positioned to undergo DSE with the previous a high-affinity binding state, depending on the tensile appendages and important subunit that is bound to the CTDs30 (FIG. 1b, step 4). forces applied to the pilus36,41–44. This affinity modulation virulence factors that are used The process of DSE occurs through a zip‑in‑zip-out mechanism enables UPEC to adapt to the flow condi- by bacteria to enable attachment, biofilm formation mechanism, whereby the P5 residue of the N-terminal tions experienced in its environment. In the absence of and both twitching and gliding extension of the incoming subunit engages the empty external forces, the adhesin–receptor interactions are motility. P5 pocket in the groove of the previous subunit. The G1 relatively weak and thus enable bacteria to disseminate (donor) strand of the chaperone, and thus the chaper- through the urinary tract, whereas in the presence of Pilins Pilus subunits, which form a one, is displaced, as the P4, P3, P2 and P1 residues of the stronger forces, such as those that are induced by urine pilus in their assembled state. N-terminal extension progressively occupy their respec- flow, UPEC can resist being flushed out. A pilus can contain several tive hydrophobic pockets in the groove of the previous thousand copies of a single subunit21,31,32. Once displaced, the chaperone is released Structure and function of the pilus rod. Recently, pilin or may be composed of into the periplasm. DSE is followed by the transloca- structures were determined for the type 1 pilus (using more than one type of pilin. In some pili, the terms ‘tip pilin’ tion of the growing pilus through the usher pore and its a hybrid scanning transmission electron microscopy 45 and ‘anchor pilin’ refer to pilins transfer to the CTDs of the usher (FIG. 1b, step 5). The (STEM) and solid state NMR approach) and for the P that are present at the tip or NTD‑to‑CTD transfer is driven by a differential affinity pilus to ~3.8 Å resolution (using cryo-­electron micros- the base of the pilus structure, of the chaperone–subunit complex for the NTD (lower copy (cryo‑EM))46; these structures showed a similar respectively. affinity) and the CTDs (higher affinity)33. In addition, overall organization. The structure of the P pilus rod in Lectin domain there is a favourable energetic ‘path or track’ that forces its coiled state shows a right-handed superhelix of 3.28 A versatile carbohydrate- the subunit in the pore lumen to rotate while it is being PapA subunits per turn, an axial rise of 7.7 Å and a heli­ binding domain found in many translocated out34. This cycle of subunit incorporation is cal pitch of 25.2 Å (FIG. 1c,d). Overall, P pili are ~81 Å Gram-negative bacterial pili then repeated to add subunits to the base of the growing in diameter and have a hollow lumen that is ~21 Å in that enables bacteria to attach FIG. 1b to host tissues during infection. pilus ( , step 6). Both the tip fibrillum and rod ele- diameter. This atomic model of the P pilus explains ments of the pili are assembled in this sequential man- the mechanism of rod uncoiling at the molecular level SecYEG translocon ner. For the P pilus, the stochastic incorporation of the by revealing the details of an extensive inter-subunit An evolutionarily conserved termination subunit PapH terminates pilus biogenesis. interaction network46. The DSE interactions that con- membrane transporter that is located in the cytoplasmic This is because PapH does not have a P5 pocket and nect adjacent subunits are strong hydrophobic inter- 35 membrane of bacteria and thus cannot undergo DSE with any other subunit . It actions that are topologically essential, as they provide archaea, and the membrane of is unclear how the biogenesis of type 1 pili stops, as a the fold-complementing interactions for each subunit the endoplasmic reticulum in termination subunit has not yet been identified. in the polymer20,21,47. By contrast, the rest of the PapA eukaryotic cells. In bacteria, subunit interaction network, which is responsible for this machinery transports proteins into the periplasm and Structure and function of the tip fibrillum. The overall maintaining the helically wound quaternary structure can insert membrane proteins architecture of type 1 pili and P pili consists of a short, of the pilus rod, is composed of predominantly weak into the inner membrane. thin tip fibrillum that is mounted on a much longer and hydrophilic contacts. Furthermore, these weak contacts thicker rod (FIG. 1a,c). The structure of the tip fibrillum are topologically non-essential, as they are not involved Donor-strand 46 complementation of type 1 pili is known both in isolation and in a com- in subunit folding and stability . Distinct sets of strong (DSC). A mechanism plex with the usher, and the two structures are simi- hydrophobic and weak hydrophilic interactions explain whereby an incomplete lar34,36. The structure of the FimH adhesin has also been how chaperone–usher pili can gradually uncoil when immunoglobulin‑like fold in a solved both pre-transport and post-transport through an external force is applied48–51: the weaker hydrophilic pilus subunit is completed and the usher. Indeed, on comparing the structure of the interactions between the subunit stacks that form the stabilized in the periplasm by a donor strand from a dedicated FimD usher bound to FimC–FimH (representing FimH pilus quaternary structure are disrupted, but polymer 30 periplasmic chaperone (either pre-transport ) with the structure of FimD bound to integrity is retained through the strong hydrophobic and FimC or PapD). the tip fibrillum (representing FimH post-transport34), topologically essential DSE interactions.

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a Chaperone–usher pathway P pilus Lectin domain PapG Pilin domain (adhesin)

Type 1 pilus PapF Tip fibrillum Lectin domain FimH (adhesin) Pilin domain PapE Tip fibrillum FimG

FimF PapK

Rod Rod FimA PapA

OM FimD (usher) PapC (usher)

Plug N-terminal extension Plug N-terminal extension CTD1 CTD1 NTD CTD2 NTD CTD2 PG FimC PapD PapH

Periplasm

IM SecYEG Translocon Bacterial cytosol

b Mechanism of translocation Plug OM 1 2 3 4 5 6

CTD1

NTD DSE CTD2 Chaperone Periplasm Usher Adhesin An outer membrane- c d embedded protein that Adhesin catalyses the assembly of chaperone–usher pili. This Crystal structure: protein is composed of a Type 1 tip fibrillum 24‑stranded β‑barrel pore domain, a periplasmic –1 amino‑terminal domain (NTD), 0 two periplasmic carboxy‑ +1 terminal domains (CTD1 and Molecular CTD2) and a plug domain. surface PapA monomer Donor-strand exchange P pilus rod in P pilus P pilus rod (DSE). A mechanism n ~ 1,000 molecular surface whereby an incomplete Cryo-EM PapA immunoglobulin‑like fold in a map 90° pilus subunit is completed and stabilized by a donor strand OM that is provided by the –1 amino‑terminal extension of +1 0 an adjacent pilus subunit. This occurs once pilus subunits are Usher assembled into the growing Periplasm pilus by the usher. Adjacent PapA subunits

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Type II secretion systems The pilus rod may contribute functionally to the bio- T4P enable bacteria to adhere to host cells and 52 (T2SSs). Large macromolecular genesis of pili. The bacterial periplasm lacks ATP , which other surfaces, aid in the formation of biofilms, bac- nanomachines present in raises the question as to what drives the trans­location of terial aggregates and microcolonies, and are involved various pathogenic and non- chaperone–usher pili. Random out- and backsliding in cellular invasion, electron transfer, phage and DNA pathogenic Gram-negative 1,6,57 bacteria that are responsible Brownian motions of pilus subunits could occur in the uptake, and twitching or gliding motility . Most T4P for the secretion of folded usher pore; backsliding could be prevented by the for- can elongate and retract through the action of several proteins (including enzymes mation of the quaternary helical rod structure at the cytoplasmic ATPases, which provides the basis for their and toxins) from the periplasm exit of the usher, which would result in pilus ­extrusion5. role in bacterial motility58,59. Furthermore, they have to the extracellular However, this hypothesis remains to be tested. remarkable biomechanical properties and can exert environment. and withstand considerable forces in excess of 100 pN Secretin Type IV pilus system (REF. 60). T4P can be classified into type IVa (T4aP) and Large, multimeric and gated Type IV pili (T4P) are surface organelles that are present type IVb (T4bP) subgroups on the basis of the features outer membrane pore-forming in many bacterial pathogens, in which they act as impor- of their major pilin protein and the organization of their proteins that are found in type 61 IV pilus systems, type II and tant virulence factors that are responsible for causing pilus genes . This section focuses on recent structural 53 type III secretion systems, and human . These dynamic extracellular append- progress for T4aP systems and, unless stated otherwise, in some filamentous ages range between 6–9 nm in diameter and can reach uses the nomenclature from Pseudomonas aeruginosa. extrusion several micrometres in length. They are found in Gram- systems. negative and Gram-positive bacteria, and in archaea6,54,55. Biogenesis of T4P. T4P systems are similar to type II In archaea, such systems are responsible for the assembly secretion systems (T2SS), which translocate folded pro- of the archaeal or ‘­’ (REF. 56). teins from the periplasm of Gram-negative bacteria into the extracellular environment6. However, in the T4P system the principal substrates for translocation are the ◀ Figure 1 | The architecture, biogenesis and structure of chaperone–usher pili. pilin subunits, which form the pilus filament (FIG. 2a). a | Architecture of type 1 (left side) and P (right side) chaperone–usher pili. Pilins are In the first step of the biogenesis of T4P, precursor transported across the inner membrane by the SecYEG pathway. In the periplasm, pilin (or prepilin) subunits, which contain a cytoplas- a chaperone (FimC or PapD) facilitates pilin folding and stabilization. The chaperone– pilin complexes are transported across the periplasm to an outer membrane-embedded mically exposed N‑terminal signal peptide, are inserted usher (FimD or PapC), at which point pilin polymerization occurs. The usher is composed into the inner membrane by the SecYEG machinery. of a 24‑stranded β‑barrel pore, a plug domain, an amino‑terminal domain (NTD) and two Once in the inner membrane, the signal peptide is carboxy‑terminal domains (CTD1 and CTD2). The pilus fibre is composed of a tip cleaved, which results in the exposure of an N‑terminal fibrillum that is capped with an adhesin and attached to a rod structure. The stochastic phenylalanine residue that is methylated by the prepi- incorporation of PapH into the P pilus terminates pilus biogenesis. b | Subunit lin peptidase PilD62,63. At this stage, the conserved and translocation through the usher is illustrated using crystal and modelled structures from hydrophobic N‑terminal α‑helix of mature pilin subunits type 1 pilus and P pilus systems. Proteins are colour-coded as in panel a. The chaperone– spans the inner membrane and the C‑terminal globular adhesin complex binds to the NTD (modelled using RCSB Protein Data Bank (PDB) domain is exposed to the periplasm1 (FIG. 2a). During entries 3BWU130 and 1QUN14) of the usher (modelled using PDB entries 3OHN and elongation of the pilus, mature pilins are extracted from 3RFZ30) (step 1). The plug domain relocates to a position next to the NTD and the chaperone–adhesin complex transfers to the CTDs. The pilin domain of the adhesin the inner membrane and are incorporated into the base interacts with the CTDs, whereas the lectin domain traverses the usher pore (PDB entry of a growing pilus by the T4P biogenesis machinery. 3RFZ30) (step 2). The next chaperone–subunit complex is recruited to the NTD and the The T4P biogenesis machinery is composed of sev- N-terminal extension of the subunit is positioned towards the hydrophobic groove of eral subcomplexes, all of which are required to form a the adhesin (modelled using PDB entries 3RFZ30 and 3BWU130) (step 3). Donor-strand functional system64 (FIG. 2a). The outer membrane secretin­ exchange (DSE) occurs; the N-terminal extension of the subunit replaces the donor subcomplex includes PilQ, a gated multimeric outer strand of the chaperone through a zip‑in‑zip-out mechanism and the chaperone is membrane pore protein through which the growing 30 130 44 recycled (modelled using PDB entries 3RFZ , 3BWU and 4XOE ) (step 4). The pilus is assembled and disassembled65–68, and the pilotin chaperone–subunit complex that was previously bound to the NTD is transferred to protein PilF69 (FIG. 2a–c). In gonorrhoeae and the CTDs, and the adhesin inside the pore translocates upwards (modelled using PDB , the T4P secretin-associated pro- entries 3RFZ30 and 4J3O34) (step 5). The cycle is repeated and the nascent pilus grows (PDB entry 4J3O34) (step 6). c | Structural model of a chaperone–usher pilus. The ~3.8 Å tein (TsaP) is also part of the secretin subcomplex and cryo-electron microscopy (cryo‑EM) map (grey) of the P pilus rod (EM Data Bank (EMDB) may anchor it to the peptidoglycan through 70 entry EMD‑3222 (REF. 46)) is positioned on top of the usher into the extracellular space. its peptidoglycan-binding LysM motif (FIG. 2a,b). The molecular surface (purple) generated from the model of the P pilus (PDB entry However, deletion of the putative TsaP homologue in 5FLU46) is mapped on top and linked to the tip fibrillum surface, as determined in the P. aeruginosa had no effect on the expression or func- crystal structure of FimD–FimC–FimF–FimG–FimH (PDB entry 4J3O34). This model is a tion of T4P67. Further work is required to define the role hybrid of structures from type 1 and P pili systems but provides a visual picture of an of this protein and its homologues in the biogenesis of entire chaperone–usher pilus. The inset shows a ribbon diagram of the P pilus rod T4P. The motor subcomplex is composed of the inner structure and highlights one PapA molecule (purple) among its neighbouring subunits membrane ‘platform’ protein PilC and the cytoplasmic (grey). d | The molecular surface of the P pilus rod (PDB entry 5FLU46) with the PapA ATPases PilB and PilT, which are responsible for pilus subunits coloured in shades of purple and green (upper panel). The central subunit 71–73 (FIG. 2a,b) (boxed) is ‘subunit 0’ and the preceding and succeeding subunit are –1 and +1, elongation and retraction, respectively . The respectively. A rotated view of the boxed region, presented as a ribbon diagram of three ‘alignment’ subcomplex, which is composed of PilM, adjacent PapA subunits in the P pilus rod, is also shown (lower panel) and highlights the PilN, PilO and PilP, bridges the secretin and the motor mechanism of DSE. IM, inner membrane; OM, outer membrane; PG, peptidoglycan. subcomplexes74–78 (FIG. 2a,b). Recently, it was shown that Part a is adapted with permission from REFS 5,46, Elsevier. Part b is adapted with the alignment subcomplex is not simply a static link permission from REF. 5, Elsevier. between the secretin and motor subcomplexes, but that

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a Type IV pilus machinery Elongation Retraction

b Myxococcus xanthus type IV pilus machinery: architectural model

PilA

Non-piliated Piliated Growing pilus PilQ PilQ Pore

OM

OM C PilQ C PilA TsaP TsaP PG N N PilP PilO PilP Periplasm PilN PilN PilA PilO PilC

PilC

IM PilM PilM Bacterial cytosol ATP PilB PilT PilB ADP ATPases Elongation Retraction

c d type IV pilus

PilE monomer in D-region type IV pilus αβ-loop C α1C

Cryo-EM map: type IV pilus P22 (Neisseria meningitidis) α1N G14 140º E5 OM N PilE monomer

PG Cryo-EM map: PilQ (secretin) (Pseudomonas Periplasm aeruginosa)

Right-handed IM four-start helix Helical core of filament Bacterial cytosol

Nature Reviews | Microbiology

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Pilotin dynamic interactions among alignment subcomplex which enables their visualization inside the cellular Proteins that function to members functionally contribute to pilus extension and environment (in situ), albeit at low resolution. This ensure correct secretin retraction79. The interaction between the alignment approach was used to visualize both the non-piliated localization, assembly and and secretin subcomplexes is through PilP and PilQ75,80. (closed) and piliated (open) states of the T4P systems outer membrane insertion. By contrast, multiple interactions contribute to the of Thermus thermophilus84 and M. xanthus85 at 32–45 Å Amidase N‑terminal bridging of the alignment and the motor subcomplexes, and 30–40 Å resolution, respectively. Overall, the T4P domains including the interaction between PilM and PilN77, and machinery in both organisms is composed of the same (AMIN domains). Domains that between PilC and the cytoplasmic ATPases72. The final core components. However, T. thermophilus lacks a are widely distributed among structural component of the T4P system is the helical PilP homologue and there are some differences in their bacterial peptidoglycan (FIG. 2d) hydrolases and transporters pilus filament , which in P. aeruginosa is com- cytoplasmic ATPases. T. thermophilus has a much larger located in the periplasm, and posed of major (PilA) and minor (FimU, PilV, PilW, PilX ­periplasmic space than M. xanthus and other Gram- are thought to interact with the and PilE) pilin subunits and adhesin molecules, such as negative bacteria; therefore, its T4P machinery has to peptidoglycan cell wall. The PilY1 (REFS 81–83). be longer to span both membranes (~70 nm versus presence of AMIN domains in 86,87 secretins is thought to help ~30 nm) ; it achieves this length mainly by having a 66 anchor the type IV pilus Structure of T4P machinery. The difficulty of puri- much longer PilQ secretin protein . machinery in the cell wall. fying intact and fully assembled complexes presents FIGURE 2b shows a hypothetical architectural model challenges in the structural investigation of large of the M. xanthus system that was constructed by fit- multi­protein assemblies. However, this limitation may ting existing structures into the densities observed in be overcome through studying multi-component com- the cryo‑ET studies of this system85. The ring-forming plexes using cryo-electron tomography (cryo‑ET), components (TsaP, PilP, PilN, PilO and PilM) of the T4P machinery were modelled in a 12‑fold stoichiometry, as this provided the ‘best fit’ with the observed cryo‑ET ◀ Figure 2 | Type IV pilus (T4P) architecture, biogenesis and structure. a | Components density85, although other stoichiometries have also that constitute the type IV pilus (T4P) system are shown. Pilins are inserted into the inner been proposed64,75. Changes that were observed in the membrane by the SecYEG translocon (not shown) and are then incorporated into the piliated state compared with the non-piliated state in base of the growing pilus. The T4P machinery is composed of the outer membrane secretin subcomplex (PilQ, TsaP; the pilotin PilF is not included in this figure), the both T4P systems from M. xanthus and T. thermophi- alignment subcomplex (PilM–PilN–PilO–PilP), and the inner membrane motor lus included: the pilus traversing the periplasm into the subcomplex (PilC–PilB–PilT). ATP hydrolysis by PilB and PilT provides energy for pilus extracellular space, the opening of the PilQ gate (there elongation and retraction, which may involve the rotation of PilC. b | The architectural is an additional gate present in the longer PilQ protein model of a type IVa pilus (T4aP) system in the non-piliated (RCSB Protein Data Bank (PDB) of T. thermophilus), and the presence of additional cyto- entry 3JC9 (REF. 85)) and piliated (PDB entry 3JC8 (REF. 85)) state in Myxococcus xanthus plasmic density that can be attributed to the elongating (the amidase amino‑terminal (AMIN) domains of PilQ have been removed for clarity). All ATPase84,85 (FIG. 2b). In addition, the distance between components are coloured-coded as in panel a. This model was constructed by fitting the inner membrane and the outer membrane is larger existing structures of T4P components into electron density maps that are derived from in M. xanthus85 (FIG. 2b), maybe due to the presence of cryo-electron tomography (cryo‑ET) experiments. TsaP has an N‑terminal LysM motif that the nascent pilus. binds to peptidoglycan and a carboxy‑terminal domain that is connected through a flexible linker. The proteins in the alignment subcomplex connect the motor subcomplex In addition to our growing understanding of the over- with the secretin subcomplex. PilN and PilO form coiled coils that link their all architecture of the T4P system, we are continually periplasmically located globular domains to the inner membrane, and the N‑terminal tail learning more about its individual components. Recently, of PilN interacts with PilM, which is located in the cytoplasm. PilP is a lipoprotein that is a ~7.4 Å cryo‑EM structure of the P. aeruginosa secretin anchored to the inner membrane. The piliated model differs as it includes the ATPase pore was determined67 (FIG. 2c). The P. aeruginosa secre- PilB, a shift of the outer membrane and the secretin pore complex, and the presence of a tin pore consists of two amidase N‑terminal domains pilus fibre that extends from the inner membrane, through PilQ and into the extracellular (AMIN domains), an N0 domain, an N1 domain and the space. In this figure, we have updated the model constructed in REF. 85 with a new secretin pore domain. The cryo‑EM structure resolved 92 atomic model of the Neisseria meningitidis PilE pilus fibre (PDB entry 5KUA ). c | The ~6 Å the secretin and N1 domains but not the N0 and AMIN cryo-electron microscopy (cryo‑EM) map (grey) of the N. meningitidis T4aP (EM Data Bank domains, which are presumably more flexible. This struc- (EMDB) entry EMD‑8287 (REF. 92)) is positioned on top of the ~7.4 Å cryo‑EM map (grey) of the Pseudomonas aeruginosa secretin PilQ (EMDB entry EMD‑8297 (REF. 67)). This hybrid ture showed a closed central gate near the outer mem- model provides an overall structure of these two essential T4P components; the brane–periplasm interface. To accommodate the passage remaining components of the T4P machinery are drawn as in panel a. The inset shows a of a 6 nm wide pilus, this central gate would need to be ribbon diagram of the T4aP structure and highlights one PilE molecule (purple) among displaced to the interior wall. In such an opened state, neighbouring subunits (grey). d | Left panel: the structure of the N. meningitidis T4aP can the PilQ secretin pore would form a large channel that is be a right-handed one‑start, right-handed four‑start or left-handed three‑start helix. ~80 Å wide at the periplasmic side, which would expand The molecular surface of the T4aP is coloured to show the right-handed four‑start helix, to ~100 Å near the displaced central gate and constrict to which is also indicated by the black arrow. Right panel: ribbon diagram of the PilE ~68 Å at the extracellular region. Interestingly, this struc- monomer. The disulfide region (D-region; orange) and αβ‑loop (blue) are important for ture suggests that the secretin channel is a homo 14‑mer. pilus interactions. The positions of residues that are involved in the loss of helical order However, other secretin structures have other symme- in the extended N terminus (G14 and P22), and pilus assembly and core stabilization (E5), are indicated. Zoomed‑in inset: a view of the helical regions that stabilize the core of tries, including the type III secretion system secretin the pilus. Proposed salt-bridge interactions between E5 and the N‑terminal amine InvG from Salmonella enterica subsp. enterica serovar 88 group, and hydrogen bonds between E5 and T2 (between neighbouring pilins), are Typhimurium (C15) and the T2SS secretin GspD from 89 indicated by dotted lines. α1C, C-terminal portion of α-helix 1; α1N, N-terminal portion E. coli and (C15) . There are several pos- of α-helix 1; C, C terminus; IM, inner membrane; N, N terminus; OM, outer membrane; sible explanations for these different secretin symme- PG, peptidoglycan. tries. For example, there might be structural differences

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between secretins of different secretion systems (for rate) from T. thermophilus also supports this model of example, T2SS secretins versus T4P secretins), or differ- ATPase-generated PilC rotation96. However, this model ent symmetries might correspond to different dynamic needs to be tested and confirmed. states that are related to function90. Conjugative type IV secretion pili T4P pilin and filament structure. The structures of Conjugation in Gram-negative bacteria is a process monomeric and polymeric T4P pilin subunits have been whereby a donor cell exchanges genetic material, such as well studied. Several T4P pilin crystal structures have or integrative conjugative elements (ICE), with been determined and show a conserved lollipop-like a recipient cell after establishing initial contact through a structure that has an extended N‑terminal α‑helix and conjugative pilus. Conjugation is the principal means by a globular C‑terminal domain81,83 (FIG. 2c,d). In the fully which horizontal gene transfer, a process that is crucial assembled T4aP, the N‑terminal α‑helices pack together for the spread of resistance genes among bac- in the centre of the filament and the C‑terminal globular terial populations, is mediated97. Conjugation in Gram- domains are oriented towards the outside of the struc- negative bacteria requires two main structures. First, a Conjugation ture. This ensures that the D‑region (disulfide region) versatile type IV secretion system (T4SS) that is responsible A mechanism of horizontal and the αβ‑loop, which are regions of the T4P pilin for the assembly of the conjugative pilus and the secretion gene transfer that involves the domain that are important for functional interactions,­ of different types of cargo, ranging from single-stranded transfer of genetic material 91,92 (FIG. 2c,d) from a donor to a recipient are solvent accessible . The assembly of T4P DNA (ssDNA), proteins or protein–DNA complexes. bacterial cell. pilins (major and minor pilins) into a pilus filament is Second, a dynamic conjugative pilus that can extend thought to be mediated by interactions between con- and retract98,99. Many types of conjugative pilus have Donor cell served hydrophobic residues in the N‑terminal α-helix been described100; however, this section will focus on the A cell that provides a of each pilin91 (FIG. 2d). In addition, a highly conserved F pilus, as it represents the best functionally and structur- conjugative genetic element, which is often a or glutamate at position 5 (Glu5) of each pilin subunit is ally characterized example. In particular, we focus on the an integrative conjugative required for pilin assembly and is thought to form a F pilus and the F‑like conjugative pilus that are encoded element, that is eventually salt bridge with the N‑terminal amine of its neighbour- by the pOX38 and pED208 plasmids (both are F plasmid mobilized to a recipient cell ing subunit93,94. Recently, an atomic model of the T4aP family members), respectively. The F plasmid encodes at some point in the bacterial life cycle. in N. meningitidis was constructed by fitting a 1.44 Å all of the components that are necessary for conjugation, pilin crystal structure (PilE) into a ~6 Å cryo‑EM vol- including the pilin, which assembles into the conjugative Integrative conjugative ume92. This resolution enabled the positioning of indi- T4SS pilus filament, and other components that consti- elements vidual PilE pilin subunits and secondary structural tute the T4SS machinery (FIG. 3). F plasmids have been (ICE). A large family of elements, but could not resolve side chain conforma- identified in a broad range of Gram-negative bacteria, chromosomally encoded mobile genetic elements tions. Nevertheless, Glu5 was found in a position that including, and not limited to, E. coli, Salmonella enterica 101 that encode a functional is consistent with the existence of a salt bridge between subsp. enterica serovar Typhi and Shigella flexneri . conjugative secretion system pilin subunits in a T4P filament. This interaction may that mediates their excision, promote the incorporation of pilin subunits into the The structure of the TraA pilin. F pili are assemblies of transfer and integration into 91–93 a recipient cell. Integrative growing pilus structure from the inner membrane . thousands of TraA pilin subunits that polymerize into a conjugative elements are Interestingly, this structure also revealed a loss of helical filament. The F pilus enables bacteria to establish also known as conjugative α‑helical order in the N‑terminal helical region of PilE cell‑to‑cell contact during the early stages of conjuga- transposons. (between the helix-breaking residues Gly14 and Pro22), tion. On contact, the dynamic F pilus retracts, presum- which may enable the filament to undergo spring-like ably by depolymerization, which, in turn, brings donor Recipient cell 92 (FIG. 2d) A cell that acquires a extensions in response to external force . and recipient cells into close proximity. The transport conjugative genetic element of ssDNA into the recipient cell through the F pilus is from a donor cell, which either A mechanism for T4P assembly. Recent progress in then initiated. Thus far, the structural details of TraA gets incorporated into the determining low-resolution structures of the fully have been elusive. However, recent cryo‑EM structures bacterial chromosome (for example, integrative assembled T4P machinery in situ, and of individual of two members of the F pilus family (encoded by the conjugative elements) or components and subcomplexes, has led to the develop- pOX38 and pED208 plasmids) have enabled the atomic remains in the bacterial ment of a model of T4P assembly. On the basis of the model of TraA to be constructed de novo in the context cytoplasm (for example, hypothetical cryo‑ET-derived architectural model of of a fully assembled pilus102 (FIG. 3a). The electron den- a plasmid). the T4P machinery in M. xanthus, a model was pro- sity of the TraA pilin structure encoded by the pED208 Type IV secretion system posed for pilus assembly, whereby PilB (stabilized at plasmid revealed an entirely α‑helical structure that (T4SS). A large macromolecular the inner membrane by PilM) hydrolyses ATP, which contains three α‑helices (α1, α2 and α3): the α3 helix nanomachine that is found in results in a rotation of the inner membrane platform is able to interact with both α1 and α2 helices, and the both Gram-positive and protein PilC, which, in turn, ‘scoops’ PilE pilus subunits loop between α2 and α3 projects towards the lumen Gram-negative bacteria, as well in some archaea. These from the inner membrane and incorporates them into of the pilus. Both N termini and C termini are located 85 102 systems have evolved to deliver an upward-translocating nascent pilus . This model is on the outside of the helical polymer (FIG. 3a, inset). DNA and protein substrates supported by the finding that PilB interacts with both into a wide range of PilM and PilC95, and by the knowledge that the related Structure of the helical filament. The cryo‑EM struc- prokaryotic and eukaryotic archaeal flagellar motor is also comprised of a rotat- ture of the pED208‑encoded F‑like pilus shows a fila- target cells, which promotes 56 the spread of antibiotic ing component . Furthermore, the recent structure of ment that has a diameter of 87 Å and an internal lumen 102 resistance and bacterial the ATPase region of PilB in complex with ATPγS (an that is 28 Å in diameter . The structure can either be pathogenesis. analogue of ATP that is hydrolysed at a much slower described as a five‑start helical filament (FIG. 3b, left

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panel) or as pentameric layers that are stacked on top whereas VirB7, VirB9 and VirB10 are channel compo- of each other related by a 28.2° rotation with an axial nents in the outer membrane. VirB10 spans the entire rise of 12.1 Å (FIG. 3b, bottom panel). Interestingly, an periplasm and is also inserted in the inner membrane additional unconnected electron density was observed (FIG. 3d). Remarkably, three ATPases, VirB4, VirB11 and in the vicinity of each TraA molecule. This density VirD4, are usually required for T4SS function, although was consistent with the head group and acyl chain of VirB11 is absent in the F system106. Recently, an almost a phospholipid, which showed that the F pilus is made complete T4SS, encoded by the R388 plasmid (also a up of a stoichiometric 1:1 complex between TraA and conjugative plasmid), was purified and visualized using a phospholipid (FIG. 3a,b). Mass spectrometry analysis electron microscopy107 (FIG. 3a). This 3.5 MDa complex is showed that the two main phosphatidylglycerol species embedded in both the inner membrane and outer mem- that are present in the pilus are phosphatidylglycerol brane, and is composed of two large subcomplexes, the 32:1 and phosphatidylglycerol 34:1, which are the two inner membrane complex (IMC) and the outer mem- most common phosphatidylglycerol species found in brane ‘core’ complex (OMC), which are linked together the bacterial cell membrane102. The phospholipid sub- by a stalk. The components of the IMC are thought to stantially alters the electrostatic potential of the pilus be present as multiples of 12 copies of VirB3 (12 copies), lumen. Without phosphatidylglycerol, the pilus lumen VirB4 (12 copies), VirB5 (12 copies), VirB6 (24 copies) is overwhelmingly electropositive, whereas the addition and VirB8 (12 copies), whereas the OMC contains 14 of phosphatidylglycerol makes the channel moderately copies of VirB7, VirB9 and VirB10 (REF. 107). The IMC electronegative102 (FIG. 3e). This feature presumably also includes the 14 transmembrane helices of VirB10. facilitates the transport of ssDNA in the pilus lumen. It is still unclear what functional role the symmetry mis- The presence of phospholipids in the structure might match between the OMC and the IMC might have in also lower the energetic barrier for the extraction or re‑­ type IV secretion. insertion of pilus subunits from, or into, the inner mem- The OMC is formed of two layers that are termed brane, thereby facilitating pilus extension or retraction, the ‘O‑layer’ (outer layer) and ‘I‑layer’ (inner layer)108,109 respectively. The lowered energy barrier may also facil- (FIG. 3a). Crystallographic analysis of the O‑layer iden- itate pilus insertion into the recipient for tified that the channel through the outer membrane is efficient cargo delivery102. formed by a helical barrel, in which each VirB10 subunit contributes a helical hairpin110. The main structural fea- Conjugative pilus biogenesis. F pilin subunits (TraA) tures of the IMC are two periplasmic arches, an inner are synthesized as pro-pilin proteins that have an unu- membrane-embedded platform and two barrel-like sually long leader peptide approximately 50 residues in structures that protrude into the cytoplasm107. Each length4,103. The nascent protein is inserted into the inner barrel-like structure is formed of a VirB4 ATPase hex- membrane with the help of TraQ by an ATP-dependent amer (FIG. 3a). This architecture of the T4SS is likely to and proton motive force (PMF)-dependent mechanism, be preserved in Gram-negative bacteria, as suggested by which is independent of the SecYEG pathway104 (FIG. 3c, the recent cryo‑ET investigation of the T4SS apparatus step 1). On insertion, the leader peptide of the pro-pilin is in Legionella spp.111. cleaved by the peptidase LepB (FIG. 3c, step 2). This results It is not known how the T4SS apparatus assembles in a mature pilin that has its N termini and C termini a conjugative pilus. However, the VirB4 ATPase is exposed to the periplasm; the α2 and α3 helices form two known to interact with the VirB2 pilin and therefore hydrophobic transmembrane segments that are inserted might have a role in mediating the extraction of the into the inner membrane, with the α2–α3 loop oriented pilins from the inner membrane112. These data suggest towards the cytoplasm103. Next, another protein, TraX, a model whereby VirB4 catalyses the extraction of the acetylates the N terminus of TraA105 (FIG. 3c, step 3). pilins into the T4SS by an ATP-dependent process. However, the N‑terminal acetylation of TraA does not A second ATPase, VirB11, was shown to be important seem to be essential for the assembly of a functional con- for pilus assembly113, but, thus far, no VirB11 homo- jugative pilus. During the processing of TraA, each pro- logue has been identified for the F plasmid-encoded tein molecule is able to specifically engage one molecule T4SS114. Therefore, the assembly of the F pilus might of phosphatidylglycerol. This stoichiometric complex is be powered solely by the activity of the VirB4 homo- extracted from the bacterial inner membrane by the T4SS logue of the F system, potentially with the help of other and is assembled into the F pilus (FIG. 3c, step 4). components of the T4SS IMC. Perhaps, the two VirB4 Assembly of the pilus is carried out by the T4SS ATPases can work in concert to extract and assemble machinery encoded by the F plasmid. Conjugative TraA–phosphatidylglycerol­ complexes from the inner T4SSs generally consist of a core set of 12 different pro- membrane into a pilus. A growing T4SS pilus polymer- teins, termed VirB1–VirB11 and VirD4 (REF. 2) (FIG. 3d). ized from the IMC would enter the OMC, which would Proton motive force VirB1 has glycosidase activity and does not form part lead to its extrusion from the . (PMF). An electrochemical of the secretion machinery itself, but assists in break- gradient across a bacterial cell ing down the peptidoglycan layer between the inner Type V pili membrane that is generated membrane and the outer membrane.VirB2 (TraA in Type V pili are important virulence factors of the oral by the transfer of protons or electrons across an energy- the F plasmid) and VirB5 are the major and minor pathogen and have func- transducing membrane by an pilus subunits, respectively. VirB3, VirB6 and VirB8 tions in bacterial adhesion, co-aggregation and bio- electron transport chain. are channel components in the inner membrane, film formation. This can lead to gingivitis and severe

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a c

α1 OM F pilus α2 Cryo-EM map α3

TraA–phospholipid complex PG TraA–phospholipid OM complex LepB Periplasm Ac

TraQ TraX ? PG IM PMF Mature TraA pilin Periplasm TraA pro-pilin Type IV secretion system 1 2 3 4 Bacterial cytosol IM

d Type IV secretion system Bacterial cytosol

Pilus

VirB2

b Five-start Phospholipid array helical assembly in the pilus strand OM VirB7

VirB10 OMC PG

VirB9

VirB6 and VirB8

IM IMC VirB3

VirB4

Bacterial cytosol VirD4 VirB11 ATPases

e 12.1 Å

28.2°

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periodontitis115,116. They are composed of a divergent with its N‑terminal lipid moiety) and transports it to superfamily of proteins (currently more than 1,800 an outer membrane assembly machinery that remains unique members) that are found in the Bacteroidetes to be identified117,122 (FIG. 4a). At the outer membrane, phylum, particularly in the class Bacterioidia117. In a trypsin-like arginine or lysine-specific proteinase, P. gingivalis, two morphologically distinct type V pilus gingipain R or gingipain K (Rgp or Kgp, respectively), types have been identified: major or long pili (which are carries out a second essential proteolytic step before 0.3–1.6 μm in length)118 and minor or short pili (which pilus polymerization122,123 (FIG. 4a–c). This cleavage are 80–120 nm in length)119. These major and minor event occurs after a conserved arginine or lysine resi- pili are encoded in similar operons composed of genes due, which results in the removal of the first β‑strand that encode structural pilins that form the pilus stalk (A1) to generate a hydrophobic groove in the NTD of (FimA in major pili and Mfa1 in minor pili), anchor- the mature protein117,120,123 (FIG. 4b,c). In addition, crystal ing pilins (FimB in major pili and Mfa2 in minor pili), structures of P. gingivalis FimA (a type V pilus subunit other subunits­ (including tip ­pilins) and regulatory that should not be confused with FimA of type 1 chap- elements116. erone–usher pili) superfamily stalk subunits revealed that the two C‑terminal β‑strands of the CTD (A1′ Type V pilus biogenesis and structure. Type V pilins and A2′) can exist in an open or closed conformation, are composed of an N‑terminal domain (NTD) and a which suggests that there is flexibility in the preced- slightly larger C‑terminal domain (CTD), each domain ing loop region117 (FIG. 4c). In the open conformation, comprises a transthyretin-like fold that contains seven a hydrophobic groove is also exposed along the length core β‑strands (arranged into two β‑sheets)117,120,121. of the CTD in which the A1′ strand would normally They are expressed in the cytoplasm as lipoprotein pre- extend the β‑sheet. Thus, with the A1 β‑strand of the cursors that contain an N‑terminal signal peptide and NTD removed and the A1′ and A2′ strands of the CTD a consensus sequence known as a ‘lipobox’ (REF. 122) in an open conformation, the hydrophobic groove (FIG. 4a,b). Pilin proteins are then transported to the extends across the entire length of the protein (includ- periplasmic side of the inner membrane by the SecYEG ing both the NTD and CTD)117 (FIG. 4c). It has been machinery, after which a conserved cysteine residue in proposed that a strand-exchange mechanism, whereby their lipobox becomes lipidated, followed by the cleav- the hydrophobic groove is complemented by a strand age of the signal peptide by a type II signal peptidase from the neighbouring subunit, enables pilus assem- (FIG. 4a,b). From the periplasmic space, these pilins are bly117,120, similarly to the biogenesis of chaperone–­usher presumably shuttled to the outer membrane through pili. Both the N-terminal and C‑terminal regions have a lipoprotein-sorting pathway, whereby a lipoprotein been proposed to act as the donor strand during this chaperone binds to the pilin (presumably by interacting process117,120. However, cysteine crosslinking exper- iments support the idea that the flexible C‑terminal A1′ and A2′ strands of the CTD function as the donor Figure 3 | F pilus architecture, biogenesis and structure. a | A hybrid structural model of ◀ in donor-strand exchange117. Tip pilins that have been the conjugative pilus from , which shows the ~3.6 Å cryo-electron microscopy (cryo‑EM) map of the F‑like pilus from the pED208 plasmid (EM Data Bank structurally characterized either lack the flexible A1′ (EMDB) entry EMD‑4042 (REF. 102)) positioned on top of the type IV secretion system (T4SS) and A2′ strands (for example, Mfa4) or use them to from the R388 plasmid (EMDB entry EMD‑2567 (REF. 107)) in the extracellular space. Inset: interact with a fused C‑terminal lectin domain (for the molecular model of the pilus subunit formed by a TraA–phospholipid complex that was example, BovFim1C), and thus function to cap the constructed from the F pilus electron density (RCSB Protein Data Bank (PDB) entry 5LEG102). pilus structure117,120. Anchor pilins at the pilus base b | Surface representation of the molecular model of the F pilus structure from panel a. are also unique because they are not processed by Left panel: a side view of the pilus in which each of the five helical strands are individually Rgp or Kgp and they retain their N‑terminal lipid coloured. Right panel: each helical strand consists of 12.8 subunits of TraA–phospholipid modification, thereby anchoring the pilus in the outer per helical turn. Bottom panel: two adjacent pentameric units are related by an axial rise of membrane117 (FIG. 4a). 12.1 Å and a 28.2° rotation. c | The mechanism of TraA maturation in the inner membrane and the assembly of a TraA–phospholipid complex into the F pilus are illustrated. TraA Common ‘threads’ between pilus types pro-pilin is transported to the inner membrane through a proton motive force (PMF)- dependent mechanism that is facilitated by TraQ (step 1). The leader peptide from the Pili provide a means for bacteria to interact with and pro-pilin is cleaved by the LepB peptidase (step 2), followed by the acetylation of the sense their environment. Although the individual sub- mature amino terminus of the TraA pilin by TraX (step 3). During maturation, each TraA units that constitute a pilus and their mode of biogenesis molecule binds to a phosphatidylglycerol family phospholipid and the protein– can be very different, several intriguing similarities exist phospholipid complex assembles into a helical filament once inside the T4SS apparatus between unrelated pili. For example, chaperone–usher (step 4). d | Architecture of the T4SS and F pilus. Components that constitute the T4SS and pili, T4P and type V pili all mediate bacterial adhe- F pilus are shown. The T4SS inner membrane complex (IMC) is composed of three ATPases sion3,115. The modular architecture of pili enables bac- (VirB4, VirB11 and VirD4) and VirB3, VirB6 and VirB8. The outer membrane complex (OMC) teria to have various different binding modules at the tip is formed of VirB7, VirB9 and VirB10, which inserts into the outer and inner membranes and or in their structures. Interestingly, lectin or lectin-like spans the periplasm. The conjugative F pilus, which protrudes from the surface of the T4SS into the extracellular environment, is composed of the VirB2 homologue TraA. The precise domains are versatile carbohydrate-interacting domains location of VirB5 is unclear and is not indicated here. e | The electrostatic potential of the that are found in many pilus types. Both chaperone– pilus lumen is shown, calculated in the absence (left) and presence (right) of the usher pili and type V pili contain distal ‘tip’ or ‘adhesin’ phosphatidylglycerol phospholipid. Blue represents positive charge and red represents subunits; these proteins can be fusions of pilin and lectin negative charge. Ac, acetylation; IM, inner membrane; OM, outer membrane; domains that enable their successful incorporation into PG, peptidoglycan. Part e is adapted with permission from REF. 102, Elsevier. pili and confer adhesive properties, respectively14,15,117.

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One of the most fascinating properties of certain pili is ‘stretch’ when they experience tensile force, by fully their ability to ‘stretch’. This biomechanical property is extending a stretch of residues in the N‑terminal tail of most pronounced in chaperone–usher pili, which can the pilin subunit92. Therefore, T4P achieve their spring- extend to more than five times their original length by like properties at the level of their secondary structure, unstacking helically arranged subunits48. T4P can also whereas chaperone–usher pili do so at the level of a Type V pilus pathway b NTD CTD SP A1 A1ʹA2ʹ Cytoplasm CTD Tip pilin C R/K NTD A1 A1ʹA2ʹ Adaptor Periplasm subunit ? C R/K A1ʹA2ʹ OM

Mature protein

c Pilus assembly mechanism Stalk pilins ? A1ʹ Closed ? CTD conformation Other factors? A2ʹ Rgp or Kgp NTD A1 A2ʹ CTD Anchor ? OM secretion OM pilin pore?

A1ʹ NTD Rgp or Kgp and ? conformational changes A1 PG CTD Lipoprotein chaperone Open conformation A2ʹ Periplasm CTD NTD Pilins NTD A1ʹ CTD Polymerization?

IM Lipidation SecYEG OM translocon NTD Bacterial cytosol Hydrophobic groove

Figure 4 | Type V pilus architecture, biogenesis and structure. lipidated on an N‑terminal cysteine and theirNature signal Reviews peptide | Microbiology (SP; grey) a | A schematic overview of the biogenesis of type V pili. Pilus subunits are is subsequently cleaved by a type II signal peptidase. The mature protein is expressed as lipoprotein precursors and are targeted to the periplasmic side generated by a second proteolytic step at the outer membrane, whereby of the inner membrane through the SecYEG machinery. A conserved the first β‑strand (A1; yellow) is removed by Rgp or Kgp (R and K are the amino‑terminal cysteine (yellow sphere) is lipidated and then the signal conserved residues after which Rgp and Kgp cleave). The C‑terminal A1′ sequence is cleaved. Next, the pilin subunits are shuttled to the outer and A2′ strands are also indicated (boxed in red). c | The strand-exchange membrane, presumably by a lipoprotein sorting pathway through mechanism of pilus assembly is illustrated using topology diagrams. Left interactions with a lipoprotein chaperone. Subunits are secreted to the panel: each pilin is composed of an N‑terminal domain (NTD) and a outer membrane through an unknown mechanism. A second proteolytic C‑terminal domain (CTD). After Rgp or Kgp cleave the A1 strand in the NTD, step that is mediated by either gingipain R (Rgp) or gingipain K (Kgp) occurs, and the A1′ and A2′ β‑strands of the CTD have switched to an open which results in the production of the mature pilin before pilus subunit conformation, a hydrophobic groove is exposed along the entire protein polymerization through a strand-exchange mechanism. Anchor pilins do (NTD and CTD; red dashed box). This hydrophobic groove can be not undergo the Rgp-dependent or Kgp-dependent cleavage of the A1 complemented by the A1′ (highlighted in red) and A2′ β‑strands of the next strand and can anchor the pilus in the outer membrane, probably via the subunit (highlighted in red and dashed; this region is disordered in most lipid modification of its N‑terminal cysteine. The carboxy‑terminal A1′ and type V pilin structures). The precise molecular mechanisms that lead to this A2′ strands of the tip pilin are not available (absent or shielded by an strand-exchange polymerization, including the timing of A1 strand additional fused domain) for further pilus extension. Tip subunits cleavage and A1′ and A2′ strand conformational changes, are not known. presumably mediate interactions between the pilus and unidentified Right panel: the polymerization of pilins leads to the assembly of type V pili. ligands. Question marks represent parts of the pathway that involve Topology diagrams of two adjacent strand-exchanged stalk pilins are drawn unknown effectors or mechanisms. b | A protein domain map of stalk pilins in the inset dashed box with their A1′ and A2′ strands highlighted. IM, inner that shows the different forms of the proteins found in the cytoplasm, membrane; OM, outer membrane; PG, peptidoglycan. Part b is adapted with periplasm or outer membrane. In the periplasm, the pilin precursors are permission from REF. 117, Elsevier.

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quaternary structure. Chaperone–usher pili and type V field of cryo‑EM have made it possible to visualize the pili are assembled by proteins that only span the outer challenging structures of large membrane-embedded membrane1,116, whereas T4P, F pili and F‑like (conjuga- complexes or entire pilus assembly systems. tive) pili are assembled from secretion systems that span Currently, chaperone–usher pili represent the both bacterial membranes2. Interestingly, chaperone– best-characterized pilus system in Gram-negative bacte- usher pili and type V pili both use a strand-­exchange ria. However, the mechanisms of other pilus biogenesis mechanism for subunit polymerization17,20,117,120, systems have not yet being elucidated. Recent models of although they are unrelated in terms of sequence and the entire T4P assembly apparatus that were derived from domain architecture. T4P, F pili and F‑like pili have cryo‑ET experiments have provided an exciting glimpse subunits that are extracted from the inner membrane of this widespread bacterial surface structure. Future and then assembled into pili by components that are efforts will address remaining questions, such as the localized in the inner membrane and the periplasm1. exact stoichiometry of the system. This, in combination Thus, T4P, F pili and F‑like pili assembly machineries with higher-resolution structures of the assembly appa- are double-membrane-spanning systems that are pow- ratus, will add to our understanding of what seems to be ered by ATP hydrolysis and require dedicated cytoplas- an intriguing assembly mechanism. The discovery that mic ATPases for pilus assembly. This enables pili to the F pilus is composed of a 1:1 stoichiometric protein–­ ­dynamically extend and retract1. phospholipid complex adds another layer of complexity to the biogenesis and function of conjugative pili. Future Conclusions and outlook studies of the structure and function of the T4SS that Pili are crucial virulence factors that are expressed by assembles this type of pilus will hopefully reveal more many and are therefore important about the mechanism of conjugative pilus biogenesis. in human . In this Review we have focused on Last, although less is known about type V pili, recent structural progress, which has provided a wealth of progress has revealed their strand-exchange mechanism information in regard to the mechanisms of pilus assem- of polymerization. Future efforts will focus on identify­ing bly and the structures and properties of pilus filaments all of the key players in the type V pili assembly pathway themselves. Crystal structures of individual compo- and on understanding the mechanisms of these pro- nents and subcomplexes have contributed substantially cesses. The exciting recent developments in the field of to our knowledge of the biogenesis and functions of pili. structural biology will undoubtedly continue shedding­ Moreover, recent hardware and software advances in the light into these fascinating bacterial systems.

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