Structural biology of bacterial secretion systems

P pili and bacterial adhesion

Type IV secretion systems

Type V secretion systems - Autotransporters

A) P pili and bacterial adhesion

Many pathogenic Gram-negative bacteria assemble on their surfaces hair-like adhesive pili that mediate microbial attachment by binding to receptors present in host tissues. P pili are adhesive organelles encoded by eleven genes in the pap (pilus associated with pyelonephritis) gene cluster found on the chromosome of uropathogenic strains of E. coli. Six genes encode structural pilus subunits, PapA, PapH, PapK, PapE, PapF, and PapG, which assemble into a heteropolymeric fiber with an adhesive tip (PapG). The binding of PapG to galabiose receptors in the human kidney is a critical event in the pathogenesis of pyelonephritis. The P pilus consists of two sub-assemblies, a thick rigid rod of PapA subunits arranged in a right-handed helical cylinder and a thin, flexible tip fibrillum extending from the distal end of the rod and composed primarily of PapE subunits. Two subunits, PapK and PapF, act as adaptors, which link the pilus rod to the base of the tip fibrillum (PapK) or the PapG adhesin to the distal end of the tip fibrillum (PapF).

The biogenesis of P pili occurs via the highly conserved chaperone-usher pathway (Click on figure at right or here for more details). Two of the genes in the pap operon, papD and papC, encode the chaperone and usher, respectively. PapD, the prototypical periplasmic chaperone, consists of two immunoglobulin-like (Ig) domains oriented in an L shape. During pilus biogenesis, PapD binds to and caps subunit surfaces involved in subunit-subunit interactions in the mature pilus, thus preventing their premature aggregation in the periplasm. The chaperone-subunit complexes are targeted to the PapC outer membrane usher, a large protein pore complex where subunits assemble in a specific order to form a pilus. The usher is thought to facilitate uncapping of chaperone-subunit complexes to allow the formation of subunit-subunit interactions.

To gain further insight into the processes of subunit capping and assembly in the chaperone-usher pathway of pilus biogenesis, we solved the structure of a complex of the subunit PapK bound to the chaperone PapD. This 2.4 A resolution structure revealed that the chaperone PapD functions by donating its G1 strand to complete the Ig fold of the PapK subunit via a mechanism termed "donor-strand complementation". The structure of the PapD-PapK complex also suggests that during pilus biogenesis every subunit completes the Ig fold of its neighbouring subunit via a mechanism termed "donor-strand exchange" (see details in Sauer et al.,1999 and also see Figure). Recently, we were able to solve the structure of the PapE subunit both in a complex with the PapD chaperone AND in a complex with the N-terminus of PapK (see details in Sauer et al., 2002). PapE is the major subunit of the tip fibrillum and PapK is the pilus subunit which assembles after PapE (see Figure). The PapK N-terminus (also called N-terminal extension) was hypothesized to complement the fold of PapE by substituting to the chaperone G1 strand during the assembly process. The structure of the PapE/PapK complex confirms this hypothesis. The structure also shows that in the pilus, the complementing strand of PapK runs anti- parallel to strand F of PapE, which is the direction opposite to that of the PapD G1 strand in the PapE-PapD complex (see details in Sauer et al., 2002). Thus, while each subunit is complemented by the chaperone to form an atypical Ig fold where the donor- strand runs antiparallel to strand F, donor-strand exchange results in a subunit where the donated strand complements the Ig fold of the subunit in a canonical fashion i.e. running antiparallel to strand F. We believe that it is this topological transition that drives pilus assembly.

Recent work has aimed to understand the details of the donor-strand exchange (DSE) reaction. Using X-ray crystallography and real-time electrospray ionisation mass spectrometry, we demonstrated that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE towards product formation. These results support a zip-in-zip-out mechanism for donor-strand exchange and a catalytic role for the usher (details of this study can be found in Remaut et al., 2006; also, for further reading on beta-strand addition in structural biology see Remaut and Waksman, 2006). This study was also instrumental in providing the molecular basis of pilus termination in the Pap system. Indeed, it has been shown some time ago that one subunit in the Pap system, PapH, is responsible for terminating pilus biogenesis. However, the reason for this was unknown. In a study published in EMBO reports (see Verger et al., 2006), we showed that PapH is unable to undergo donor-strand exchange. By solving the structure of PapH bound to the chaperone PapD, we found that PapH has no P5 pocket. We believe this is the reason why PapH cannot undergo the exchange reaction: without an initiating point (the P5 pocket) the reaction cannot proceed and thus, when PapH is incorporated in the pilus, the chaperone bound to it cannot be challenged by any other complex.

In a recent breakthrough, we have determined the structure of the translocator domain of the PapC usher. In collaboration with David Thanassi and Huilin Li, we have determined the cryo-EM structure of the usher FimD bound to the nascent FimFGH pilus tip (see Remaut et al., 2008). These structures are the first of a translocating machinery bound to a secretion intermediate. The PapC translocator domain comprises 24 beta-strands and is occluded by a folded plug domain, likely gated by a conformationally-constrained beta-hairpin (click here for a picture of the PapC translocator domain). In the FimD complex structure, the usher is dimeric. Remarkably, the FimFGH fiber traverses only one of the two usher protomers, demonstrating that, although an usher dimer is required for function, only one usher protomer is used for secretion. A model for pilus assembly and secretion is proposed whereby the two usher protomers cooperate in chaperone-subunit complex recruitment while only one pore is used for secretion.

Finally, we have started in collaboration with Professor Almqvist at Umea University (Sweden) and our long-term collaborator, Professor Scott Hultgren (Washington University Medical School, St Louis, USA), a drug design effort which has recently culminated in the publication of the first so-called "pilicide", small molecule inhibitors of pilus biogenesis (see details in Pinkner et al., 2006). For further structural work on pilus biogenesis, see Verger et al., 2007 and Verger et al., 2008.

Pathogenicity in gram-negative bacteria is critically dependent upon secretion machineries which mediate the transport and injection of toxic molecules into target cells. These secretion systems are classified into four types (I to IV) and share a common requirement for proteins that utilize ATP as an energy source to drive transport of macromolecules.

Type IV secretion systems (T4SS) are used not only to transport molecules toxic to host cells, but also are used to transport DNA or protein-DNA complexes. One such process is bacterial conjugation whereby two mating bacteria exchange genetic material. By facilitating conjugative transfer, type IV secretion machineries play crucial roles in the spread of antibiotic resistance genes among bacteria. Type IV secretion systems have been involved in pathogenicity caused by bacteria such as of Helicobacter pylori responsible for gastric ulcers or Legionella neumophila responsible for Legionnaire disease.

The VirB/VirD system of Agrobacterium tumefaciens has served as a prototype for T4SSs. In A. tumefaciens, the T4SS is responsible for secreting the virulence factors that lead to the formation of crown galls in infected plants. This system is commonly used to generate genetically modified plants. The A. tumefaciens T4SS is composed of 11 proteins encoded by the virB operon and at least one protein, VirD4, encoded by the virD operon. Some T4SS members contain a complete set of proteins similar to the A. tumefaciens VirB/D proteins, while others do not. The figure at right (or click here) provides a schematic representation of what a T4SS may look like and where the various VirB/VirD components may locate within the secretion machinery.

Type IV secretion systems require a subclass of ATPases which are known under the generic name of VirB11 ATPases. VirB11 ATPases are essential for conjugative transfer of DNA in most systems studied to date, and toxin transfer. Beside the fact that VirB11 ATPases are known to be essential for type IV secretion and pathogenicity, little is known about the function that these proteins fulfill in these machineries. Their role may vary from one organism to another depending on the type of transport they help mediating. For example, in the conjugative system encoded by the RP4 plasmid, the VirB11 ATPase, TrbB, is known to be involved in pilus biogenesis. Indeed, many type IV secretion systems, notably those involved in conjugative transfer, function in conjunction with a fibrous cell surface organelle called "pilus" which is thought to be important for adhesion between bacteria or between bacteria and host eukaryotic cells. In Helicobacter pylori (the causative agent of ulcers), however, there is no known pilus associated with the type IV secretion system. As a result, the VirB11 ATPase of H. pylori may be directly involved in transport of toxins through the inner membrane. In either case, the potential roles of VirB11 proteins are consistent with evidence that these proteins are at least in part associated with the inner membrane (IM) and localized to the cytoplasmic side of the IM.

We have solved the crystal structure of a binary complex of the VirB11 ATPase from H.pylori bound to ADP at a resolution of 2.5 Angstrom. VirB11 is an hexameric protein. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings which together form a chamber open on one side and closed on the other. The open side of the chamber is formed by the N-terminal domain ring and has an inner-dimension of 50 Angstrom in diameter. This ring is believed to be partly embedded in the membrane. The closed side is formed by the C-terminal domains, has a conical shape shrinking to 10 Angstrom in diameter, and is believed to be exposed to the cytoplasm. Overall, the hexameric VirB11 protein has the shape of a closed grapple where the claws of the grapple, formed by the C-terminal domains, are coming together to form the base of the chamber. The structure has features reminiscent of proteins such as p97 or NSF involved in assembly/disassemly of the vesicle fusion apparatus in eukaroytes and suggests a similar role for VirB11 in assembling/disassembling the type IV secretion machinery. (See details in Yeo et al.,2000 , Savvides et al., 2003, and Hare et al., 2006; also click here to visualize the VirB11 structure). In a collaboration with Hybrigenics, we also identified the first regulator of the HP0525 ATPases, HP1451. By solving the structure of the HP0525:HP1451 complex, we determined the mode of action of HP1451 and showed that it prevents opening of the HP0525 N-terminal domains, thereby inhibiting its ATPase activity (see detais in Hare et al., 2007).

We have also solved the VirB5 structure. VirB5 is a minor component of the pilus in both the A. tumefaciens and pKM101 plasmid T4SSs (click here for location of VirB5 within the T4SS machinery). In Agrobacterium, VirB5 co-fractionates with extracellular VirB2, the major pilus component, and also with VirB7, an outer-membrane protein. VirB5/TraC is a single domain protein, which consists of a three helix bundle and a loose globular appendage (click here for visualization of the structure). Structure-based site-directed mutagenesis followed by functional studies indicates that VirB5 proteins participate in protein-protein interactions important for pilus assembly and function (for more details, please see Yeo et al (2003)). We have crystallized and solve the structures of two periplasmic components of the T4SS: VirB8 and VirB10. The structures of VirB8 and ComB10 resemble known folds, albeit with novel secondary-structure modifications unique to and conserved within their respective families. Both proteins crystallized as dimers, providing detailed predictions about their self-associations. These structures make a substantial contribution to the repertoire of T4SS component structures and will serve as springboards for future functional and protein-protein interaction studies using knowledge-based, site-directed and deletion mutagenesis (for more details, please see Terradot et al (2005)).

Recently, we have solved the NMR structure of the first type IV secretion components complex, that of the C-terminal domain of VirB9 (VirB9-CT) of pKM101 bound to its cognate lipoprotein, VirB7. VirB9-CT adopts a beta-sandwich fold. One edge of the fold constitutes the VirB7-binding site, while the other consists of a loose beta-hairpin, clearly separate from the body of the fold. Introduction of a FLAG epitope into the hairpin loop results in surface exposure, providing evidence that this part of the structure crosses the outer-membrane (see details in Bayliss et al., 2007).

Finally, we have determined the high resolution cryo-EM structure of the core complex of the type 4 secretion system encoded by the pKM101 conjugation plasmid Fronzes et al., 2009). This core complex is formed by 14-mer of 3 proteins, VirB7, VirB9, and VirB10. These proteins assemble to form a double-membrane spanning, double-walled channel consisting of two layers, the I and O layer. This is the first view of a type 4 secretion channel.

For list of type IV secretion component expression clones, see Clone list

C) Type V secretion systems - Autotransporters

Proteins secreted by the type V pathway are referred to as autotransporters. Typically, autotransporters are expressed as precursor proteins with three basic functional domains, including an N-terminal signal peptide, an internal passenger domain, and a C-terminal translocator domain (the beta-domain). The signal peptide directs export of the precursor protein across the inner membrane via the Sec machinery and is then cleaved by signal peptidase I. Subsequently, the beta-domain inserts into the outer membrane and forms a beta-barrel structure with a central channel, allowing extrusion of the passenger domain across the membrane. Once on the surface of the organism, the passenger domain is usually cleaved from the translocator domain and released extracellularly. In some cases, the passenger domain is cleaved but remains cell associated.

Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia belongs to the trimeric autotransporter subfamily. Recently, we have solved the crystal structure of the C-terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992-1098) (for more details, please see Meng et al (2006)). This domain forms a beta-barrel with 12 transmembrane beta-strands, including four strands from each subunit (click here for visualization of the structure or see at right). The beta_barrel has a central channel of 1.8nm in diameter that is traversed by three N-terminal alpha-helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three alpha-helices and the loop region between the alpha_helices and the neighboring beta-strands are essential for stability of the trimeric structure of the translocator domain and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters.