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Nat Rev Microbiol. Author manuscript; available in PMC Jul 9, 2013.
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The type II secretion system: biogenesis, molecular architecture and mechanism


Many Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner membrane platform of the T2SS is the nexus of the system and interacts with the periplasmic filamentous pseudopilus, the dodecameric outer membrane complex and a cytoplasmic secretion ATPase to orchestrate the secretion process. Here recent structural and biochemical information is reviewed to describe our current insights of the biogenesis, architecture and mechanism of the T2SS.


A wide range of systems has been discovered in the last several decades which enable bacteria to export proteins across one or two membranes into the extracellular milieu1. Many Gram-negative bacteria use the type II secretion system (T2SS) to translocate folded proteins from the periplasm, through the outer membrane, into the extracellular milieu. The T2SS is important for pathogenic and non-pathogenic species alike. Human pathogens with one or more T2SS include Vibrio cholerae2, enterotoxigenic and enterohemorrhagic Escherichia coli (ETEC and EHEC, respectively) 3-5, Pseudomonas aeruginosa6, 7, Klebsiella spp.8, 9, Legionella pneumophila10 and Yersinia enterocolitica11. Aeromonas hydrophila, a pathogen of fish and amphibia, also contains a T2SS12. Plant pathogens containing a T2SS include Dickeya dadantii (formerly called Erwinia chrysanthemi), the causative agent of fire blight; Erwinia carotovora, which is responsible for soft rot disease in potato and other crops13, Xanthomonas campestris which causes black rot in crusifers, and others14. Non-pathogens with a T2SS gene cluster include metal reducing bacteria like Shewanella oneidensis15. It has also become apparent that the T2SS, the type IV pilus system (T4PS), the archaeal flagella and the transformation system are evolutionary related and share several structural and functional features16-19 (Box 1).

Box 1

T2SS, T4PS, archaeal flagella, and transformation system of Gram-positive bacteria: related outer membrane machineries

The four systems are shown in the Figure, with related proteins in the same color: the ATPase in gold; inner membrane protein with multiple TM helices in salmon; inner membrane proteins with a single TM helix in different shades of green; outer membrane proteins in blue; pilins, pseudopilins and flagellins in purple. The pseudopilins, pilins and flagellins in all four systems are processed by a related inner membrane peptidase (not shown). The T2SS occurs in Gram-negative bacteria, the T4PS in both Gram negative and Gram-positive bacteria138. The similarities between the T2SS and the T4PS have been recognized early on. Indeed, the systems have similar (i) outer membrane conduits – secretins; (ii) filamentous structures – pseudopilus and pilus, respectively; (iii) prepilin peptidases; (iv) cytoplasmic ATPases; and (v) homologous multiple-spanning membrane proteins, GspF in the T2SS and PilC in the T4PS. The similarities between other proteins of the inner membrane platform have only become apparent when crystal structures of several domains were solved111, 116, 139. Moreover, it has been shown that T4PSs in some cases function as secretion systems. Interestingly, the archaeal flagellum has more commonalities with the T4PS and T2SS than with the bacterial flagellum18. Archaeal flagellins are processed by a preflagellin peptidase, an aspartic membrane protease that belongs to the same class as the prepilin peptidase. After cleavage by the preflagellin peptidase, the flagellin subunits are assembled into the flagellum. Almost all archaea lack an outer membrane, therefore a secretin is lacking in this system. Similarly, the transformation system that occurs in many Gram-positive bacteria has no secretin. The cytoplasmic ATPase, the inner membrane protein with multiple TM helices, the pilin/pseudopilin/flagellin, and a specific membrane protease (not shown) are common components of these four systems.

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The T2SS is a sophisticated multi-protein machinery containing 12–15 different proteins (Table 1; Fig. 1) which are generally encoded on a single operon20. The T2SS is also the main terminal branch of the general secretory pathway (Gsp)21. Hence, a Gsp prefix, followed by a capital letter, has been suggested for proteins names in all T2SSs, but this nomenclature is not universally employed. Here, a Gsp-based nomenclature is used, with the species-and-system-specific name in superscript (Table 1).

Figure 1
Type II secretion system subassemblies and biogenesis
Table 1
Composition and species-specific nomenclature1 of the T2SS.

It has been suggested that the T2S apparatus spans both the inner and outer membranes, although the fully assembled machinery has not been visualized yet in a purified form and perhaps may never be captured for analysis as it is likely dynamic in nature. Four T2SS sub-assemblies can be distinguished: the pseudopilus, the outer membrane complex, the inner membrane platform and the secretion ATPase. The pseudopilus is a periplasmic fibrous structure formed by five different pseudopilins with multiple copies of the major pseudopilin22-24. These proteins received their name due to N-terminal sequence homology to type IV pilins and their dependence on prepilin peptidase. The outer membrane complex is mainly formed by a multimeric protein known as secretin25-27. The inner membrane platform contains multiple copies of at least four core membrane proteins28, 29 and although the cytoplasmic secretion ATPase is tightly associated with the inner membrane complex, it will be considered here as a separate subassembly. The inner membrane platform has a central role in the T2SS mechanism of action since it communicates with all other elements of the system. For instance, the inner membrane platform is responsible for contacting the outer membrane complex in the periplasm, the ATPase in the cytoplasm and the major pseudopilin. The inner membrane complex might have a key role in converting conformational changes in the ATPase due to ATP hydrolysis into an extension of the pseudopilus which possibly acts as a piston that pushes exoproteins through the outer membrane channel.

In this review we will focus on the biogenesis of the T2SS subassemblies and the entire machinery, the architecture of the fully assembled system and the mechanism of T2SS action. We try to integrate the large number of currently known three-dimensional structures (Supplementary Table 1) with recent biochemical studies to illustrate our current understanding of the type II secretion system.

Biogenesis of the T2SS

During T2SS biogenesis, approximately 40-70 proteins of 12-15 different types have to come together in the final machinery. Owing to this complexity, many questions regarding the biogenesis of the T2SS are still unanswered, yet, recent results make it possible to outline several of the steps involved (Fig. 1).

Pseudopilus biogenesis

Studies on P. aeruginosa GspGXcpT and K. oxytoca GspGPulG have shown that the major pseudopilin GspG uses the Sec translocon for insertion into the inner membrane30, 31. Insertion occurs co-translationally through the signal recognition particle (SRP) pathway, which recognizes the N-terminus of GspG and targets it to the Sec translocon30, 31. Based on the sequence similarity in the N-terminal sequences among all five pseudopilins, the insertion of the minor pseudopilins GspH, GspI, GspJ, and GspK also likely involves the SRP pathway.

The region of N-terminal amino acid sequence homology between pilins and pseudopilins includes a short positively charged sequence followed by a stretch of hydrophobic residues. The segment with the positively charged residues is cleaved after transport by a specific aspartic protease called prepilin peptidase6, 32, a bifunctional enzyme that also methylates the N-terminal residue after proteolytic processing33. The gene for this peptidase may occur in the T2SS operon or somewhere else in the genome. Interestingly, in some species the same prepilin peptidase is shared by the T2SS and the T4PS as it processes both pilins and pseudopilins34. The prepilin peptidase contains 8 putative transmembrane helices35 and the aspartic acid residues that have been implicated in proteolysis function are located in the cytoplasmic loops, consistent with the location of the cleavage site in pseudopilins after transport30, 31. The crystal structure of the homologous Methanococcus maripaludis preflagellin peptidase FlaK has been recently solved36, but details of the catalytic mechanism are still not completely elucidated, especially for the bifunctional T2SS/T4PS prepilin peptidases. There are no data at the moment whether or not the prepilin peptidase is an integral part of the T2SS machinery.

After cleavage by the prepilin peptidase, the pseudopilins may reside transiently in the inner membrane with the hydrophilic domain facing the periplasmic compartment30, 31. Apart from GspK, which was not initially recognized as a pseudopilin37, most pseudopilins and type IV pilins have a glutamic acid residue in the +5 position that is thought to be involved in interactions with the N-terminal amino group of a neighbouring subunit38-40. The replacement of Glu5 in GspK by an aliphatic or Thr side chain in all species is probably related to assembly or packing requirements, but remains to be established. GspK assembles into a trimer with the pseudopilins GspI and GspJ and this trimer is likely the tip of the pseudopilus39, 41. When and how GspH41 and multiple copies of GspG pseudopilins are added to the putative pseudopilus tip also remains an open question.

Outer membrane complex biogenesis

The major outer membrane protein GspD, often referred to as secretin, belongs to a family of multimeric channels that also include secretins from the type III secretion system, the T4PS and the filamentous phage assembly system27, 42-46. The T2SS secretins carry an N-terminal signal sequence that targets these proteins to the periplasm via the Sec pathway. Many bacterial outer membrane proteins rely on the β-barrel assembly machinery (BAM) for efficient insertion into the outer membrane47, 48. However, the multimerization and outer membrane insertion of the K. oxytoca T2SS secretin GspDPulD does not seem to depend on the BAM complex49, although it should be noted that dependence on BAM proteins has been shown for the homologous N. meningitidis T4PS secretin PilQ47. In some cases the T2SS secretins rely on a small lipoprotein known as GspS (commonly referred to as pilotin), for targeting to the outer membrane50, 51. The pilotins interact with the C-terminal S-domain of the secretins50, 52. Recent studies on K. oxytoca show that the S-domain of GspDPulD is disordered but adopts a folded structure on binding to its pilotin GspSPulS 53. In order to reach the outer membrane, the pilotin itself uses the Lol pathway54.

Intriguingly, some species do not contain a gene encoding a GspS homolog. In a few of these cases, the secretins themselves are lipoproteins and therefore do not depend on pilotins for correct outer membrane targeting. For instance, a recently identified secretin GspDHxcQ from the second P. aeruginosa T2SS has a long N-terminal extension and a lipidated N-terminal Cys. The extension presumably connects the lipid group in the outer membrane to the N0 domain, which is usually the N-terminal domain of T2SS secretins, in the periplasm55. The importance of secretin lipidation, however, varies among species. On the one hand, a non-lipidated mutant form of GspDHxcQ is not functional, while lipid modification is not necessary for function of the X. campestris GspDXpsD 56. In other cases it is not immediately clear how the secretins are targeted to the outer membrane in the absence of GspS and/or direct secretin lipidation. There may be genes encoding functional homologs of GspS that are not related in sequence to the known GspS somewhere else in the genomes of these bacteria, or the secretins in these species may use a different mechanism for assembly and targeting. In fact, other proteins in addition to pilotins have been implicated in assembly of the secretin multimer in the outer membrane. For example, the peptidoglycan-binding complex ExeA-ExeB of A. hydrophila supports GspDExeD oligomerization, but its requirement can be bypassed through overexpression of GspDExeD 57-59. Moreover, a soluble periplasmic protein has recently been identified that is important for targeting the P. aeruginosa secretin GspDXcpQ to the outer membrane60.

Biogenesis of the secretion ATPase and the inner membrane platform

The cytoplasmic T2SS ATPase GspE belongs to the Type II/IV secretion ATPases61. GspE is a Zn-containing protein, different from most other Type II/IV secretion ATPases62-64. T4SS and T4aPS retraction ATPases readily form hexamers65, 66. By contrast, the evidence for the hexameric nature of GspE or T4aPS assembly ATPases has so far been mainly indirect67, 68. Whether inner membrane components or other factors are needed for proper GspE assembly remains to be determined, although it has been established that cytoplasmic membrane association of GspE requires the inner membrane protein GspL69.

The inner membrane platform proteins GspC, GspL and GspM span the membrane once, whereas GspF has three TM helices29. Each protein likely uses the Sec machinery for membrane insertion. Several studies have shown that GspC, GspL and GspM protect each other from proteolysis through protein-protein interactions70-73. Whether the different proteins assemble spontaneously into the inner membrane platform, or instead require interactions with the pseudopilus and/or the ATPase, remains to be established. It has been suggested, however, that the outer membrane complex supports the assembly of the inner membrane platform as GspC and GspM form fluorescent foci in the V. cholerae cell envelope when fused to the green fluorescent protein in the presence but not absence of GspD73.

Combining subassemblies

Limited information is available regarding how and when the subassemblies interact with each other. The cytoplasmic secretion ATPase might be permanently associated with the inner membrane platform69; however, when the various pseudopilins start interacting with the inner membrane platform remains a mystery as does the number of pseudopilins associated with the inner membrane complex prior to the encounter of an exoprotein. An interaction between the major pseudopilin GspGEpsG and the inner membrane protein GspLEpsL has been reported and might have a key role in biogenesis of the pseudopilus74. The outer membrane complex contacts the inner membrane platform through GspC51, 75-78, and there are suggestions that the outer membrane complex might be involved in the assembly of the inner membrane platform73. This leads to a possible biogenesis pathway for the T2S machinery: Firstly, pilotins and the Lol system enable the formation of the secretin multimer in the outer membrane (Fig. 1a), while processed pseudopilins are accumulating in the inner membrane with their hydrophilic domain facing the periplasm. The secretin assists the formation of the inner membrane platform73 and associated ATPase (Fig. 1b) while the inner membrane platform might assemble minor pseudopilins into the tip of the pseudopilus (Fig. 1c). At some point, several major pseudopilin subunits are added from below to the tip to extend the pseudopilus, specifically by GspL with help of ATP hydrolysis by the secretion ATPase GspE. The addition of GspH and multiple pseudopilin GspG subunits to an initial GspKIJ or Gsp KIJH tip might possibly occur after an exoprotein interacts with the outer membrane complex.

Architecture of the T2SS subassemblies

The pseudopilus

The three-dimensional structures of all five pseudopilins exhibit a remarkable diversity in their globular domains that nonetheless share several common features; a long N-terminal α-helix followed by a variable region and a quasi-conserved β-sheet (Fig. 2a). The first half of the N-terminal α-helix is hydrophobic, although all pseudopilins except GspK contain a glutamate residue at the +5 position. Three crystal structures of GspG homologs reveal a Ca2+ binding site that seems to be present throughout the major pseudopilin family despite differences in coordinating aspartate residues79. Substitution of the coordinating residues in the Ca2+ site in V. cholerae GspGEpsG impairs the function of the T2SS79, perhaps by affecting subunit contacts in the pseudopilus or interactions with other T2SS proteins. The minor pseudopilins GspH and GspJ have more extended variable regions, whereas GspI and GspK have a single β-strand in the variable region (Fig. 2a)39, 80-82. These differences might serve as determinants of the pseudopilus assembly order. Interestingly, GspK possesses a unique α-domain composed from two structurally similar units39. Additionally, the α-domain of GspK contains an essential disulfide bond83 and has a binuclear Ca2+ binding site of unknown function formed by several conserved amino acid residues.

Figure 2
Structures of the T2SS pseudopilins

The crystal structure of the ETEC GspK•GspI•GspJ heterotrimer39 revealed a right-handed pseudohelical subunit arrangement that had been previously inferred from the GspI•GspJ dimer structure81 (Fig. 2b). In the heterotrimer, the subunits form a triangular arrangement with GspK at the top, followed by GspI and GspJ. The N-terminal α-helices of the three pseudopilins interact at the centre of the trimer, resembling the interactions in models of the T2SS pseudopilus and the T4P pilus (see below). GspK is likely the tip of the pseudopilus since its large globular domain extends above the smaller globular domains of GspI and GspJ39 (Fig. 2b). This is consistent with a role of GspK in preventing pseudopilus extension beyond the cell envelope. Indeed, P. aeruginosa GspKXcpX deletion mutants display longer surface exposed fibres, whereas strains with elevated levels of GspKXcpX display shorter fibres when GspGXcpT is artificially overproduced84, 85. In vitro studies of P. aeruginosa pseudopilins have shown that GspHXcpU interacts with the GspKXcpX•GspIXcpV•GspJXcpW trimer41. Therefore, it has been suggested that GspI acts as an initiator of pseudopilus assembly by binding GspJ and GspK, followed by GspH recruitment41. This suggestion is in agreement with the finding that production of surface exposed pseudopili when GspGPulG is overproduced is severely hampered in a mutant where the gene encoding GspIPulI had been deleted22. Furthermore, it has recently been shown that K. oxytoca GspIPulI•GspJPulJ self-assemble in the membrane, recruit GspKPulK and initiate pseudopilus assembly165.

The GspK•GspI•GspJ crystal structure and the T4aP PilE electron microscopy-based pilus model share several common features including the right-handed nature of the fibre with the central arrangement of the N-terminal helices, a ~10 Å shift between subunits along the fibre axis and a ~100° rotation about the fibre axis38, 39. Therefore, it has been suggested that the T2SS pseudopilus might assemble with the same helical parameters as the T4aP pilus79, 80. By contrast, a second T2SS pseudopilus model with different helical parameters has been suggested based on earlier electron microscopy data from fibres obtained by GspGPulG overproduction and a novel molecular modeling procedure40, 86, 87. In this model, the pseudopilin subunits are arranged in a right-handed fibre with a 10 Å helical rise, similar to the T4aP pilus model, but with a different subunit-to-subunit helical turn of 84.5° 40, 87. The model has been tested using double-cysteine substitutions in the hydrophobic region of the N-terminal helix followed by cross-linking and charge-altering substitutions in putative salt-bridges40. However, the substitutions in the hydrophobic region of the N-terminal helix lie close to the centre of the fibre and might be relatively insensitive indicators of the helical turn angle. Higher resolution electron microscopy data or a crystal structure of a complex of several consecutive pseudopilin subunits might establish the T2SS pseudopilus structure. Alternatively, the pseudopilus could adopt several conformations depending on the functional state, similar to type IV pili and type I fimbriae88-90.

Architecture of the outer-membrane complex

The major T2SS outer membrane protein, the secretin GspD, forms dodecamers25, 26. Individual T2SS secretin subunits have a modular multi-domain nature, with a conserved C-terminal domain and four N-terminal domains27. The structure of an ETEC secretin GspD fragment, which includes the N-terminal N0, N1 and N2 domains, has been solved using nanobodies as crystallization chaperones91 (Fig. 3a). The structure revealed two lobes, one composed of the N0-N1 domains and the other by the N2 domain, connected by a potentially flexible linker. The N0 domain adopts a fold also seen in the signaling domain of the TonB-dependent receptor92, the L. pneumophila type IV secretion system protein DotD93, the E. coli type VI secretion system protein VgrG94 and the T4 bacteriophage protein gp2795. The N1 and N2 domains share the hnRNP K homology (KH)-fold96 which is also adopted by the N3 domain according to sequence similarities. Although a high resolution structure of the C-terminal domain of GspD is still lacking, it is thought to form a mostly β-strand assembly in the outer membrane25, 97.

Figure 3
Structures of the T2SS secretin and pilotin

The structure of the V. cholerae secretin GspDEpsD was determined at 19 Å resolution using cryo-electron microscopy (Fig. 3b)26. The cylindrical structure with 12-fold symmetric features has a shape of an inverted cup, a diameter of 155 Å and length of 200 Å. The surface of the outer membrane domain seems relatively smooth, whereas the surface of the periplasmic domain is segmented with three distinguishable concentric rings. The cross-section of the GspD channel reveals a large periplasmic vestibule and a smaller extracellular chamber separated by a continuous periplasmic gate. The periplasmic vestibule has a wide opening of 70 Å that narrows down to a 55 Å constriction approximately two-thirds of the way into the channel. By contrast, the extracellular chamber is closed from the top by an extracellular gate that has a small opening of 10 Å.

The N0-N1-N2 crystal structures of GspD could be mapped into density of the wall of the periplasmic chamber (Fig. 3b) 26. A dodecameric ring of N0-N1 domains fits into the wider bottom part of the wall, whereas rings of the N2 and N3 domains can be accommodated above. According to this model, the N3 domain ring occupies the constriction site of the wall and may have a role in initiating conformational changes during protein secretion. Despite the differences in sequence, symmetry and biological function, secretins from other transport systems share common features that include the periplasmic gate formed by a part of the secretin domain and a constriction site possibly formed by the N3 domain27.

Pilotins are essential for secretin biogenesis in some species. The crystal structure of the putative EHEC T2SS pilotin GspS (PDB 3SOL) reveals an arrangement of 4 α-helices with helix 4 bending around helix 1 and a stabilizing disulfide bond (Fig. 3c), as has also been observed in K. oxytoca GspSPulS 163. Despite its stabilizing function, the intramolecular disulfide seems to be dispensable for functioning of the pilotin GspSPulS 83. The groove formed by helices 1, 3 and 4 hints to a possible secretin binding site. Notably, the structure of GspS is not related to the previously determined structures of T3SS and T4PS pilotins98-102. Whether the pilotin is part of the final T2SS machinery or disengages after the secretin is multimerized and inserted into the outer membrane remain unclear, although in some instances the pilotin has also been shown to be a component of the assembled outer membrane complex103.

Secretion ATPase

All T2SSs have a single ATPase named GspE that is thought to provide the mechanical force for the secretion process. GspE contains Walker A and B motifs, that are essential for secretion and ATPase activity9, 64, 69, 104, 105. The crystal structure of N-terminally truncated V. cholerae GspEEpsE revealed a two domain fold consisting of the N2 and C domains of the ATPase63 (Fig. 4a), the latter of which contains a Zn2+ ion coordinated by a tetracysteine motif64. The Zn-binding motif is also present in the T4aPS extension ATPase PilB, but not in other secretion ATPases. Interestingly, N-terminally truncated GspEEpsE formed a helical filament with 61 symmetry in the crystal, which suggests an oligomeric nature of the protein. Indeed, several homologous secretion ATPases have been shown to form hexamers which can adopt diverse regular and irregular arrangements of six subunits in a ring65, 66, 106-109. This is also evident in models of V. cholerae GspEEpsE based on the structure of the P. aeruginosa T4aPS retraction ATPase, PilT66, 109 (Fig. 4a). Although purified GspEEpsE is mostly a monomer, a higher molecular mass oligomer with increased ATPase activity is also present in small amounts64, 67. Site directed mutagenesis studies of interface residues in the putative GspEEpsE hexamer further support the suggestion that the functional form of GspE is hexameric68. Interestingly, the ATPase activity of V. cholerae GspEEpsE is greatly increased by the cytoplasmic domain of GspLEpsL and acidic phospholipids67.

Figure 4
Structures and topologies of the secretion ATPase and the inner membrane components of the T2SS

Two crystal structures of X. campestris N0-N1-GspEXpsE have been elucidated 110. In both structures the N1 domain adopts the same helical fold as the N1 domain of V. cholerae GspEEpsE 111, 112. The extra N-terminal domain, called N0-GspE, only occurs in a subfamily of the T2SS ATPases, e.g. X. campestris XpsE, and appears to be able to adopt different conformations110.

The architecture of the inner membrane platform

The inner membrane platform of the T2SS is composed of multiple copies of at least four different membrane proteins: GspM, GspL, GspF and GspC. GspM has a short cytoplasmic sequence, a trans-membrane helix (TMH) and a periplasmic domain (Fig. 4b). The crystal structure of the periplasmic domain of V. cholerae GspMEpsM consists of two αββ repeats forming a circular permutation of the ferredoxin fold113. Interestingly, there is an extra, peptide-like, electron density in the cleft between two subunits of a dimer in the structure, indicating that GspM might bind a partner protein at this site.

GspL consists of a cytosolic domain, a TMH and a periplasmic domain (Fig. 4b). The crystal structure of the cytoplasmic domain of V. cholerae GspLEpsL revealed distant homology to the actin-like ATPase family111, 114. However, no ATPase binding site has been found in the structure. This is in contrast to a T4PS protein, Thermus thermophilus PilM, which is homologous to the cytoplasmic domain of GspL, and has a site that is capable of binding ATP115. Remarkably, the chain of the periplasmic domain of V. parahaemolyticus GspLEpsL adopts the same circular permutation of the ferredoxin fold observed in the periplasmic domain of GspMEpsM (Fig. 4b)116. In the crystals, two of these periplasmic domains of GspLEpsL form a dimer with an extensive interface. However, the site of interaction in the periplasmic GspLEpsL dimer differs from that of the GspMEpsM dimer.

GspF is the only polytopic inner membrane protein of the T2SS with three TMHs, and has two homologous cytoplasmic domains with ~ 28% sequence identity116 (Fig. 4b). The N-terminal cytoplasmic domain of V. cholerae GspFEpsF consists of a bundle of six anti-parallel helices117. In the crystals, the N-terminal domains form a dimer with two Ca2+ binding sites in the interface. However, the metal coordinating residues are not well conserved, and the physiological relevance of metal binding is not yet established.

GspC consists of a short cytoplasmic segment, a TMH, and two periplasmic domains, the homology region (HR) domain and a PDZ domain (Fig. 4b). In some GspC homologs, the PDZ domain is absent or replaced by a coiled-coil domain118. The structure of the PDZ domain of V. cholerae GspCEpsC reveals a circular permutation of the canonical PDZ domain76. The peptide-binding groove in the PDZ domain of GspC appears to be wider than in other PDZ domain structures and might be able to accommodate an α-helical binding moiety rather than a peptide in an extended conformation 76. Although the PDZ and coiled-coil domains can be swapped without loss of function119, in D. dadantii the deletion of the PDZ domain abolishes secretion of all proteins except one, hence the PDZ domain might be involved in regulation of secretion specificity120.

The recent crystal structures of the HR domain of ETEC GspC in complex with the N0-N1 domains of GspD, showed that the HR domain is composed of six β-strands forming two anti-parallel β-sheets (Figs. 3a, ,4b)4b) 78. The surface of this domain is highly irregular with respect to charge distribution and contains a deep pocket of still unknown function.

The global T2SS architecture

Interactions of the inner membrane platform and the secretion ATPase

The cytoplasmic domain of V. cholerae GspLEpsL contains a cleft between domains II and III which forms the binding site for the N1 domain of the ATPase GspEEpsE 111. This extensive interface confirms the role of GspL as a tethering protein for the recruitment of ATPase to the inner membrane platform69. This interface is conserved across many species, suggesting that the motions that the ATPase may undergo during ATP hydrolysis (Fig. 4a) might be conveyed to the rest of the secretion system by GspL68.

Although the T2SS ATPase GspE is likely a hexamer (Fig. 4b), little direct evidence is available for the number of inner membrane proteins in the T2SS machinery. It has been speculated78, however, that an equal number of subunits of GspL, GspM and GspC might occur. In addition, a 1:1 ratio of GspE and GspL is likely on the basis of the crystal structure where one N1 domain of GspE interacts with one cytoplasmic domain of GspL111. Taking these observations together, GspE, GspL, GspM and GspC might be present in an equimolar ratio in the inner membrane platform. And since GspE is probably a hexamer, there would then be six subunits of each of these four proteins. The number of subunits of GspF, a major inner membrane protein with multiple predicted transmembrane helices, remains unknown.

Intriguingly, the crystal structures of the first cytoplasmic domain of GspF, the cytoplasmic domain of GspM, the cytoplasmic domain of GspL, and the periplasmic domain of GspL all contain dimers with C2 symmetry111, 113, 114, 116. Combining dimers of GpsL, GspM and GspF with a hexamer of GspE with C6 symmetry is non-trivial however116. It is therefore possible that one, some, or all of the GspM, GspL and GspF dimers seen in four crystal structures represent an intermediate state in the assembly process, rather than dimers which occur in the assembled T2SS. Alternatively, some of the dimers seen in the crystals might occur transiently when secreting folded exoproteins.

Interactions of the inner membrane platform and the pseudopilus

Although genetic data suggest that the major pseudopilin GspG interacts with the ATPase GspE121, biochemical and cross-linking studies have shown that GspG interacts with the inner membrane platform protein GspL74. Since GspL also interacts with the secretion ATPase GspE, the latter authors suggested that GspL has a crucial role in the conversion of energy from ATP hydrolysis into the assembly, or at least the elongation, of the pseudopilus. Although the site of interaction remains to be identified, processing of GspG by prepilin peptidase is a prerequisite for GspG-GspL interaction44. Minor pseudopilins may also associate with GspL as truncated forms of D. dadantii GspJOutJ and GspLOutL were found to interact in the yeast-two-hybrid system122.

Interactions of the inner membrane platform and the outer membrane complex

The periplasmic HR domain of GspC has been implicated in binding to the secretin GspD75-77, 123. A recent crystal structure of the complex between the HR domain of ETEC GspC and the N0-N1 fragment of the secretin GspD shows that the HR domain interacts with the N0 domain of the secretin (Fig. 3a) 78. Furthermore, the functional relevance of the interface has been confirmed by analysis of interface substitutions in the bacterial two-hybrid system, in vivo functional assay and fluorescent localization studies in V. cholerae78. Recent Surface Plasmon Resonance (SPR) studies indicate that in P. aeruginosa the N3 domain of GspDXcpQ is needed for the interaction between the periplasmic domains of GspDXcpQ and GspCXcpP 123. The periplasmic domains of GspCXcpP consist of an HR domain followed by a predicted coiled coil domain. The latter has no sequence similarity with the PDZ domain that follows the HR domain of GspC in most other species, including ETEC and V. cholerae76. The importance of the N3 domain for the periplasmic GspDXcpQ–GspCXcpP interaction in P. aeruginosa might reflect a difference between species due to the unique domain structure of GspCXcpP. Alternatively, GspCXcpP may bind to the N0 domain of GspDXcpQ but the N3 domain stabilizes the complex by either providing a second site of interaction or by inducing a conformational change in the N0 domain to strengthen the interaction.

The first crystal structure of a complex between domains from an outer and an inner membrane protein of the T2SS78, raises questions regarding the stoichiometry of the T2SS. The 1:1 complex of GspD and GspC (Fig. 3a), combined with electron microscopy evidence that GspD forms a dodecamer25, 26, suggests that there might be 12 copies of GspC interacting with the dodecameric ring of GpsD domains in the periplasm. However, when a dodecameric ring model of N0-N1-GspD domains, based on the electron microscopy reconstruction of V. cholerae GspDEpsD, is combined with twelve copies of the ETEC N0-N1-GspD•HR-GspC complex, slight clashes occur between the HR-GspC domains. Possibly, minor adjustment in either the N0-N1 ring or the HR-ring might alleviate these clashes. Alternatively, the dodecameric ring of periplasmic GspD might only have space for alternating GspC-HR domains, resulting in six GspC molecules per T2SS78.

The latter option would be compatible with the hexameric nature of the secretion ATPase, and suggests that there would be six copies of each of GpsC, GspL, GspM and GspE in the inner membrane platform (with the number of copies of GspF still unknown). This arrangement might have an approximate overall cyclic C6 symmetry for the combined inner and outer membrane complex. The helical symmetry of the pseudopilus does not follow this possible C6 symmetry and clearly the organization of the entire T2SS still has numerous unanswered questions.

Mechanism of T2SS exoprotein secretion

Initially, exoproteins are synthesized with N-terminal signal peptides that target them for cytoplasmic membrane translocation through either the SecYEG or Tat complexes124. Following removal of the signal peptides and release from the cytoplasmic membrane the exoproteins transiently reside in the periplasmic compartment prior to outer membrane translocation as shown by pulse-chase experiments125. The periplasmic intermediate is generally stable and remains secretion competent even under conditions which uncouple the cytoplasmic and outer membrane translocation events126. Our current understanding of the mechanism by which the T2SS functions remains limited. The prevalent idea is that exoprotein binding to periplasmic domains of GspD, GspC and/or the pseudopilus tip, stimulates ATPase activity of GspE, followed by adding pseudopilins subunits to the pseudopilus. The growing pseudopilus then functions as a piston pushing exoproteins through the secretin channel16, 51 (Fig. 5). Recent electron microscopy studies in V. cholerae on exoprotein binding by the secretin GspDEpsD 127 and SPR studies in P. aeruginosa on exoprotein interactions with periplasmic domains of GspDXcpQ and GspCXcpP as well as with the components of the pseudopilus tip123, respectively, support this model. Numerous aspects of this model still need to be confirmed, in particular the structure and dynamics of the inner membrane platform and the structure of the open outer membrane channel. Growth and in particular retraction of the pseudopilus are other key aspects of the model still to be confirmed or disproved. Retraction might not be well orchestrated, however, given e.g. the absence of a second ATPase which in other systems is responsible for retraction128.

Figure 5
Possible mode of action of the T2SS

Several studies have shown that the T2SS translocates exoproteins in a folded form across the outer membrane125, 129-132. Using mass spectrometry approaches, the secretomes of L. pneumophila, E. carotovora and V. cholerae have been reported recently133-135 but the T2SS secretion signal remains a mystery136. Exoproteins tend to be rich in β-strands137 and this is the case when looking at currently available structures of T2SS-secreted exoproteins (BOX 2).

Box 2

Characteristics of T2SS exoproteins

The T2SS is capable of transporting a wide range of substrates. Although it may only secrete a single protein in some species it transports between 15 (P. aeruginosa and E. carotovora) to more than twenty different proteins (V. cholerae and L. pneumophila) in other species20. These proteins have a range of biological functions, but they are generally enzymes. They include proteases, lipases, phosphatases and several enzymes that process complex carbohydrates, and with the exception of toxins, which act inside eukaryotic cells, their site of action is primarily extracellular. Folding into a secretion competent conformation in the periplasm seems to be a prerequisite for secretion125, 129-132. What this conformation presents to the T2SS for successful recognition and transport is presently unknown, but the secretion competent conformation may be identical or very similar to the conformation of the fully secreted proteins as it was recently shown that active LasB from P. aeruginosa can bind to GspC, GspD and the tip components of the pseudopilus123. Many studies, most often using chimeric proteins with easily detectable reporter proteins, have identified domains or regions that carry information for secretion; however, these domains are relatively large and have not been narrowed down to a common secretion signal140-144. The ability of some reporter proteins to contribute to the secretion of chimeras has also complicated the interpretation of some results. The secretion motif may even consist of residues from different parts of the protein and be generated only after folding144 or assembly, as in the case of oligomeric toxins125, has taken place. Although the crystal structures for a limited set of substrates suggest that a relatively β-strand rich content may be common to proteins that are transported through the T2SS (see examples in Figure Box 2), the T2S signal has yet to be identified. The displayed structures are: V. cholerae AB5 cholera toxin145 (PDB ID 1S5E), Vibrio harvey chitinase A146 – which has 82% sequence identity to V. cholerae chitinase (PDB ID 3B8S), V. cholerae neuraminidase147 (PDB ID 1W0P), P. aeruginosa elastase148 (PDB ID 1EZM), P. aeruginosa exotoxin A149 (PDB ID 1IKQ), A. hydrophila proaerolysin150 (PDB ID 1PRE), Burkholderia glumae lipase–chaperone complex151 with the chaperone highlighted in blue (PDB ID 2ES4), D. dadantii pectate lyase PelC152 (PDB ID 2PEC), D. dadantii pectate lyase PelI153 (PDB ID 3B4N), D. dadantii cellulase Cel5154 (PDB ID 1EGZ), Klebsiella pneumoniae pullulanase155 – which has 92% identity to K. oxytoca pullulanase (PDB ID 2FHB). Several of these secreted proteins contain disulfide bridges which are formed in the periplasm131, 156. The lipase–chaperone complex151 is so far a unique example of a chaperone involved in the T2SS. Several exoproteins, like cholera toxin, are multimeric proteins. Others, like pullulanase, are large single chain proteins. How this wide range of proteins is recognized by the T2SS as substrates and subsequently translocated across the outer membrane is a fascinating outstanding question.

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The similarity in structure of the N0 domain of GspD and the signaling domain of the TonB-dependent outer membrane receptor FpvA from P. aeruginosa, combined with the fact that a β-strand of the signaling domain binds to a β-strand from a region elsewhere in the structure of FpvA, led to the suggestion that the exposed strand β2 of the N0 domain might be involved in exoprotein binding as well as in interacting with other T2SS proteins91. In the recent crystal structure of the HR-domain of GspC interacting with the N0 domain of GspD, strand β2 of the N0 domain of GspD is also accessible for potential interactions with exoproteins78. Although not revealing a precise T2SS secretion signal yet, these observations suggest that β-strands complementation might be important in early steps in the secretion process and that the N0 domain of GspD and the HR domain of GspC might be key players in the initial stages of exoprotein binding.

Final conclusions

Despite considerable recent progress regarding the structural biology of the T2SS, there are still major gaps in our understanding of its biogenesis and architecture. In particular, the inner membrane complex, the nexus of interactions with the other components of the system, is still poorly understood. The same holds for interactions with exoproteins. Structural studies are required to reveal the nature of these interactions where the variation in function and structure of the exoproteins is intriguing (See Box 2). In addition, the substantial conformational changes which need to occur in GspD to open the periplasmic gate and enlarge the extracellular gate to allow the folded exoproteins to reach the extracellular milieu, remain to be clarified. The inner membrane platform likely has to change conformations in various places and moments during the secretion process. For example, the inner membrane platform is most likely responsible for the transfer of a signal to the ATPase that exoprotein binding has been directly or indirectly sensed by GspC, and, subsequently, for converting the conformational changes of the ATPase into a pseudopilus extension process. Most puzzling are the steps leading to disassembly/retraction of the pseudopilus after expulsion of the exoprotein. The possibility exists that in the T2SS, where no retraction mechanism is required to import molecules, a rather disorderly disassembly of the pseudopilus might occur after which most pseudopilins end up in the outer leaflet of the inner membrane, ready to be used for a next pseudopilus extension event.

Supplementary Material

Supplementary information S1


We like to thank the many members of our groups who have made important contributions to the studies reported. Space does not allow to mention all names but special thanks go to Jan Abendroth, Mark Robien, Stewart Turley, Tanya Johnson, Marcella Patrick and Miranda Gray. We also thank our many collaborators on the T2SS project, including Jan Steyaert and Els Pardon for the preparation of valuable nanobodies, and Tamir Gonen and his group for electron microscopy studies. This work was supported by awards RO1AI049294 (to M.S.) and RO1AI34501 (to W.G.J.H.) from the National Institute of Allergy and Infectious Diseases, while also earlier support from HHMI to WGJH is deeply appreciated.


General Secretory Pathway (Gsp)
A traditional name for the type II secretion system in which substrates are transported through the inner membrane via Sec or Tat pathways. Historically, the T2SS was discovered to rely on the Sec pathway, hence the name general. However, many more systems utilize the Sec pathway, and the term Gsp is considered less accurate by some authors
Signal Recognition Particle (SRP)
A universally conserved protein-RNA complex involved in targeting of secreted proteins to the Sec translocon for co-translational transport. Prokaryotic SRP is formed by the Ffh protein and 4.5S RNA
Sec translocon
A universal pathway for transport of proteins through the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes. The prokaryotic Sec translocon is composed of the SecY-SecE-SecG integral membrane channel, the peripheral membrane ATPase SecA and the auxiliary release proteins SecD and SecF. Targeting to the Sec translocon relies on the SRP for co-translational transport or on the cytoplasmic chaperone SecB for post-translational transport
Tat (twin-arginine translocation) pathway
A system for transport of proteins in a folded form via the cytoplasmic membrane of bacteria and archaea and thylakoid membrane of plant chloroplast. A conserved twin-arginine motif is present in the N-terminal signal sequence of substrate proteins
Beta-barrel assembly machinery (BAM)
A machinery for correct folding and insertion of the outer membrane (β-barrel) proteins
Lol pathway
A machinery for transport of outer membrane lipoproteins from inner membrane to outer membrane. Lol pathway consists of the ABC (ATP-binding cassette) transporter LolCDE, the periplasmic chaperone LolA and the outer membrane receptor/release assistant LolB
The smallest antigen-binding fragments of heavy-chain-only antibodies present in camelids. Nanobodies have a single immunoglobulin fold domain and three antigen-binding loops
Crystallization chaperones
Proteins selected to bind a particular target to use in co-crystallization. Examples of crystallization chaperones include antibody fragments and designed scaffold proteins. Crystallization chaperones may reduce conformational heterogeneity of the target protein and/or form additional crystal contacts
Walker A and B motifs
The Walker A motif, also known as P-loop (phosphate-binding loop), is a pattern GXXXGK(T/S) found in many nucleotide binding proteins. The Walker A motif interacts with phosphate groups of the bound nucleotide. The Walker B motif is a pattern XXXXD, in which X denotes any hydrophobic residue. The Walker B motif coordinates magnesium ions and is essential for ATP hydrolysis
Ferredoxin fold
A common protein fold with βαββαβ secondary structure
PDZ domain
A ubiquitous protein domain of approximately 90 amino acids that is typically involved in protein-protein interactions or signaling. Commonly found in eukaryotic proteins, but relatively rare in bacterial proteins. The PDZ acronym is derived from the first identified proteins that shared this domain: Post-synaptic density protein PSD-95, Drosophila septate junction protein Disk-large and the epithelial tight junction protein ZO-1
Circular permutation
A change in the protein sequence that leads to a similar three-dimensional structure but with a different connectivity


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