Pili and Fimbriae of Gram-Negative Bacteria

Chapter 8

Pili and Fimbriae of Gram-Negative Bacteria Ender Volkan1,2, Vasilios Kalas1 and Scott Hultgren1 1

Washington University School of Medicine, St Louis, MI, USA, 2Cyprus International University, Nicosia, North Cyprus

INTRODUCTION Pili, also known as fimbriae, are filamentous, polymeric, rigid, protein appendages assembled by Gram-negative and Gram-positive bacteria. Their discovery was first made by electron microscopy, which revealed thin fibrous, non-flagellar appendages on bacterial cells [1]. Later, two scientists, Duguid (1955) and Brinton (1965), named these non-flagellar structures fimbriae (plural, thread in Latin) and pili (plural, hair in Latin), respectively. Duguid saw Escherichia coli cells with thin appendages agglutinate red blood cells [2], whereas Brinton observed the ability of thin, fibrous F pili in transferring genetic material between two bacterial cells [3]. Hence thereafter, the terms pili and fimbriae have been used interchangeably to describe proteinaceous, non-flagellar bacterial surface filaments. Several types of pili assembled by Gram-negative and Gram-positive bacteria are involved in a range of functions involving, but not limited to, adhesion, survival, niche adaptation and spread. Certain types of pili function as receptors for bacteriophages [4 8], participate in conjugation for DNA uptake and transfer [9 11], or confer bacterial gliding and twitching motility [12 15]. Other types of surface fibres, such as curli, are amyloid fibres thought to promote biofilm formation, as well as mediating adhesion to host proteins [16 20]. Adhesive pilus fibres mediate the interaction between pathogens and specific host cell-surface ligands that allow a pathogen to establish a foothold in a particular host tissue [21,22].

CHAPERONE USHER PATHWAY PILI Arguably, one of the most important roles bacterial pili have in disease is the ability to mediate binding with host Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00008-1 © 2015 Elsevier Ltd. All rights reserved.

surfaces. One of the major pathways involved in assembly of adhesive pili in Gram-negative bacteria is the chaperoneusher pathway (CUP). CUP pili are thin, hair-like, unbranched surface extensions involved in adherence and biofilm formation [23]. These linear structures are composed of thousands of 12 20-kDa pilus subunits. CUP pili are assembled at the outer membrane of Gram-negative bacteria by two proteins: a periplasmic chaperone and an outermembrane protein called the usher. Chaperones of the CUP systems provide the scaffold for correct and efficient folding of subunits. The usher is involved in recruitment, catalysis and polymerization of chaperone subunit complexes into a mature pilus and the translocation of the growing pilus to the extracellular milieu. One of the first observations of CUP pili function was likely in 1908 when Guyot reported bacterial-mediated haemagglutination [24]. Since 1908, significant improvements and discoveries have been made in the field of CUP pili revealing unique structural, biophysical and biochemical phenomena of protein folding and assembly. Studies of CUP pili have exposed their crucial functions and essentiality in pathogenesis. For instance, the importance of CUP pili in diseases like cystitis (type 1 pili) [22,25 28], neonatal meningitis (S pili) [29,30], bubonic plague (F1 antigen) [31 33] and pyelonephritis (P pili) [34 37] has been demonstrated. For example, type 1 pilus-mediated attachment to bladder epithelium is a crucial step in initiating cystitis [21,22]. Similarly, P pili, assembled by several Gram-negative bacterial species such as uropathogenic Escherichia coli (UPEC), bind to Galα (1 4)Gal sugar moieties on human kidney for initial colonization that leads to the establishment of pyelonephritis [36,37]. CUP pili are abundant among different pilus structures assembled by Gram-negative bacteria. CUP genes were

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discovered in almost all species of the family Enterobacteriaceae (Escherichia coli, Salmonella spp., Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus spp.) in addition to bacteria belonging to other genera such as Pseudomonas spp., Haemophilus spp., Burkholderia spp., Acinetobacter spp. and Bordetella spp. [38]. Considering the prevalence of CUP pili in Gramnegative bacteria, their role in infection, and the homology they all share in terms of structure and assembly, studying CUP pili is crucial to understanding disease and adhesion by Gram-negative bacteria. Historically, CUP systems have provided prevailing models for analysis and understanding of folding, binding, assembly and structural aspects of a wide range of virulence-associated proteins. Here, we will focus on the structure and assembly mechanism of P and type 1 pili to review established mechanics of CUP pilus assembly.

Caf1M chaperone, gives rise to the entire extracellular part of the pilus [49]. Even under high magnification, individual fibres are not clearly visible as F1 antigen covers the cell surface to form a gelatinous antiphagocytic capsule B2 nm thick [49 51].

CUP PILUS ARCHITECTURE

ASSEMBLY PROTEINS AND MECHANISMS

CUP pili consist of several pilus subunits assembled into linear, fibrillar structures. The morphology of CUP pili ranges from fibrous capsule-like pilus structures (Caf1 pili from Yersinia pestis) to thick helical rods topped with thinner fibrillar tip structures (P pili from UPEC) [38]. P and type 1 pili are composite bipartite structures consisting of a tip and a rod component. The P pilus adhesin, PapG, tips the fibre followed in order by subunits PapF, PapE, PapK, PapA and PapH. PapF functions as the tip adaptor, connecting the adhesin to PapE, the subunit that forms the bulk of the tip fibre [39,40]. PapE is connected to the rod via PapK, the rod adaptor subunit. The rod, which comprises the bulk of the pilus, is made up of PapA subunits. Over 1000 PapA copies homopolymerize to establish the rod into a right-handed, helical structure. The PapA rod is 6.8 nm in width and 2.5 nm in pitch, where about 3.3 PapA subunits stack per turn [41,42]. PapH is the last subunit to be incorporated, a dual-function pilin which terminates pilus assembly while anchoring the mature pilus to the bacterial outer membrane [43,44]. Type 1 pili have a similar, bipartite architecture with a slightly more compact overall structure [45]. The tip fibrillum is made up of a single copy of the adhesin FimH [46], after which a single copy of the tip subunit FimG connects the 10 19-nm tip to the rod via FimF, the adaptor subunit [45]. The rod is composed of over 1000 copies of the major pilin subunit, FimA. Type 1 pilus termination and anchoring mechanisms remain unclear; however, recent studies suggest that FimI may function as the terminator [47,48] (Fig. 8.1). Examples of different CUP architectures exist. For instance, unlike type 1 and P pili, the Caf1 system from Y. pestis forms a capsular type F1 antigen structure, where a single subunit (Caf1), assembled by Caf1A usher and

Periplasmic Chaperones

SUBUNIT STRUCTURE Pilin subunits have Ig-like folds. However, pilin subunits lack a seventh β-strand, the G β-strand, and thus are incomplete Ig folds. Pilus subunits lack the necessary information to fold since the missing G β-strand creates a hydrophobic groove (P1 P5 pocket) that, when exposed, is detrimental to the stability of subunits in the periplasmic space [52]. Thus, subunit folding depends on a periplasmic chaperone, which transiently provides the missing information necessary for folding (see next section).

Both eukaryotes and prokaryotes use specialized posttranslational assembly proteins called chaperones to assist with macromolecule biogenesis [53,54]. The pilus subunits have to first translocate across the inner membrane, cross the periplasmic space, and then localize to the outer membrane. In Enterobacteriaceae, pilus assembly systems like the CUP require dedicated periplasmic chaperone proteins for macromolecular assembly. Bacterial periplasmic CUP chaperones such as FimC (type 1 pili) and PapD (P pili) (Fig. 8.1) are required to facilitate the folding of pilus subunits into conformations that provide stability and proper assembly into macromolecular structures. The crystal structure of the PapD chaperone solved by Holmgren and Branden [55] inspired decades worth of work on the mechanism of action of PapD and other PapD-like chaperones, which led to breakthroughs in their role in infection and in the design of antivirulence therapeutics. The PapD crystal structure revealed a boomerang-shaped protein made up of two globular domains, whose topologies are identical to that of an Ig-like fold. Interestingly, the C-terminal of PapD shares significant homology with CD4, the HIV receptor [56].

The Chaperone Necessity CUP chaperones provide steric information required for the folding of subunits and shielding of subunit interactive surfaces to keep subunits in an assembly-competent state. In the absence of CUP chaperones, pilus assembly does not occur due to misfolding and degradation of pilin subunits in the periplasmic space [52,57 59]. While bacteria lose the ability to assemble pili in the absence of the

Chapter | 8 Pili and Fimbriae of Gram-Negative Bacteria 149

FIGURE 8.1 Operon and structural organization of P and type 1 pili. A graphic depicting pili from the P (top left) and type 1 (top right) systems. The operon organization of each pilus is also shown.

chaperone, several SOS responses are activated within bacterial cells to counteract the stress created by misfolded subunits. The Cpx two-component system is one of the responders of OFF-pathway subunits, inducing a variety of genes encoding periplasmic protein folding factors like the oxidoreductase DsbA, prolyl isomerases and periplasmic proteases like DegP (Fig. 8.2). DegP resides in the periplasmic space as a hexamer in the absence of periplasmic stress conditions. Upon substrate binding, DegP assembles into a functionally active 12-mer or 24-mer [60,61]. Activated DegP degrades misfolded pilins, preventing their toxic buildup in the periplasm. DsbA catalyses disulphide bond formation in proteins required for the assembly of P pili. PapD itself is largely misfolded in the absence of DsbA, unlike the chaperone of the type 1 pilus system, FimC. This is due to the presence of a nonconserved disulphide bond in PapD that is required for efficient PapD folding [62]. Thus, DsbA mutants are severely attenuated for the production of P pili due to

misfolded PapD. Furthermore, DsbA impacts disulphide bond formation within the subunits as well. For instance, in the absence of DsbA, the adhesin of the P pilus system, PapG, does not form disulphide bonds properly [62]. When PapD is not in complex with its cognate subunits, it can form dimers to shield its surfaces from unfavourable interactions [63]. The same phenomenon of homodimerization was also observed with SfaE, the chaperone of S pili [64]. Interestingly, the CUP chaperone from Yersinia pestis, Caf1M, forms tetramers as part of a self-capping mechanism to prevent aggregation [65].

Conserved Chaperone Surfaces and Donor Strand Complementation PapD-like chaperones of CUP systems share significant homology in sequence and structure [66]. CUP chaperones have conserved interactive surfaces that are essential

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FIGURE 8.2 Current model of pilus assembly. The inner-membrane SecYEG machinery transports the pilus subunits into the periplasmic space using their secretion signal. In the periplasmic space, the subunits mediate binding with the chaperone, which facilitates their folding and stability. DsbA and prolyl isomerase are needed for proper folding of the chaperone subunit complexes. Unfolded subunits go through degradation by the DegP protease. The chaperone subunit complexes are then targeted to the assembly platform, the usher, which lies gated at the outer membrane. Upon chaperone adhesin binding to NTD (cyan), the usher gets activated to assemble pili. The plug domain (navy) swings open to bind with the NTD, such that the NTD plug complex becomes involved in chaperone subunit recruitment. The CTD2 domain of the usher (purple) catalytically dissociates the chaperone adhesin from the NTD to dock on the CTDs (purple and yellow). The transfer of the chaperone subunits about the usher domains continues until the arrival of the chaperone terminator complex, which binds to the plug NTD complex. Unable to transfer to the CTDs or go through DSE, this domain ends pilus biogenesis, anchoring the pilus to the cell surface.

for carrying out their functions. For instance, the active site of the PapD chaperone resides at the cleft between its two domains. Two conserved residues, R8 and K112, found at this cleft are essential for subunit binding and pilus biogenesis [67,68], as they form salt bridges with the C-termini of subunits [67]. In addition to R8 and K112, alternating hydrophobic residues L107, I105 and L103, located in the seventh (G1) β-strand of domain 1 of PapD, are needed for efficient subunit binding (Fig. 8.3) [63]. Due to the incomplete Ig fold of the subunits, they lack all of the necessary information to fold. The chaperone uses its hydrophobic residues L107, I105 and L103 to transiently donate its G1 β-strand in a process termed donor strand complementation (DSC) to complete transiently the Ig fold of the subunit by providing the missing G β-strand. Upon formation of the salt bridge between subunit and residues R8 and K112, the chaperone docks hydrophobic residues that lie on its G1 strand to the P1 P4 pockets of the subunit in register, leaving the P5 pocket open [57,58]. The G1 strand of the chaperone is donated in trans in a non-canonical, parallel fashion to facilitate folding of the subunits. DSC thereby provides the secondary structural element missing from the subunit [69,70] (Fig. 8.3). Mutating residue I105 is particularly

detrimental to pilus biogenesis [71]. However, mutagenesis of residue L107 does not alter pilus assembly, which may be due to the plasticity of the P1 pocket as revealed by molecular dynamics [72]. Another set of conserved, hydrophobic, solventexposed residues are found opposite to the subunit binding surface on the N-terminal domain of the PapD-like chaperones [66]. In PapD, these residues are L32, Q34, T53, P54, P55, V56, R68 and I93 (termed Set B residues), and mutating these residues interferes with pilus assembly but not with binding to and stabilization of the subunits (Volkan & Hultgren unpublished data) [66]. Corresponding residues in FimC were shown to be involved in binding to the usher’s N-terminal domain by X-ray crystallography studies [73]. Interestingly, rationally designed pilicide compounds that inhibit pilus biogenesis were shown to target Set B residues in PapD [74]. In the presence of pilicides, chaperone subunit complexes were unable to bind the usher, indicating the importance of Set B in usher binding [74]. To support these findings, biolayer interferometry studies carried out on the Set B residue point mutant P55A prevented chaperone adhesin interaction with the N-terminal domain of the usher (Volkan & Hultgren, unpublished data), further elucidating this surface as an N-terminal

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FIGURE 8.3 Chaperone conserved residues and subunit binding. (a) Chaperone PapD (green) shown in complex with the pilin domain of the PapGII adhesin (magenta) (PDB ID 3MEO). The conserved residues found on the chaperone are highlighted where Set B residues are coloured cyan (L32, Q34, T53, P54, P55, V56, R68, I93) and specific Set C residues are coloured red (D81, R82, E83, S84). The chaperone’s G1 strand used in DSC is shown in navy blue. The G1 strand’s hydrophobic residues used in DSC are shown in yellow (L103, I105, L107) while its basic residues (K112, R8) involved in hydrogen bonding with the subunit are shown in orange. (b) Donor strand complemented PapGII pilin domain (magenta). The chaperone donates its G1 β-strand (navy blue) in a parallel, non-canonical fashion where its hydrophobic residues (L103, I105, L107; yellow) complement the hydrophobic groove of the subunit while the basic residues (R8, L112; orange) are located further down and are involved in hydrogen bonding with the C-terminal strand of the subunit.

usher-targeting region. Similarly, mutating residue I93 to an alanine also completely abolished binding of the PapD PapG complex to the usher NTD, as determined by co-precipitation assays (Volkan & Hultgren, unpublished data). These studies altogether strengthen the findings that the usher NTD-targeting site resides within the conserved Set B residues of the chaperone. Another set of residues, termed Set C (L78, P79, D81, R82, E83, S84), is located at the elbow region of the protein. Set C residues are highly conserved/invariant and surface-exposed (particularly P79, D81 and R82) but have no known direct interacting partners. These residues are also part of a highly conserved DRES motif (D81, R82, E83, S84) (Fig. 8.3). The residues of this motif mediate a charge charge/hydrogen bonding network at the domain interface, which involves a salt bridge interaction with residues D196 and R116 [71]. This network of interactions is thought to be important for orienting the domains with respect to each other [75]. Considering the surfaceexposed nature of some of these residues, it is conceivable that any interactions mediated by these residues and the usher and/or subunits may cause a conformational change in the chaperone that thereby disrupts the hydrogen bonding/salt bridge network, triggering a domain reorientation. This may further allow a release of the chaperone. A similar DREA motif found in Ig-like

cadherin molecules is thought to participate in such a cascade of interactions, leading to domain reorientations and domain domain assembly interactions.

Donor Strand Exchange The chaperone, which brings the folding competent subunits to the usher, is replaced at the periplasmic side of the usher with the upcoming subunit. This occurs between the chaperone’s G1 strand and the N-terminal extension (Nte) of the next subunit via a concerted exchange mechanism called donor strand exchange (DSE). By a zip-in, zip-out mechanism, the Nte is inserted into the unoccupied P5 pocket of the groove in the previously assembled subunit [76]. Thereafter, the incoming subunit’s Nte replaces the chaperone’s G1 strand in a stepwise progression from P5 to P1 pockets. DSE, while establishing a canonical, stable interaction between the two subunits, also helps remove the chaperone from the subunit due to the thermodynamically favourable interaction between subunits. This reaction is repeated for every chaperone subunit complex until the terminator arrives at the usher. The terminator subunit of P pili, PapH, has a blocked P5 pocket, which precludes its ability to propagate DSE, thus terminating pilus assembly.

152 PART | 1 Bacterial Structure

USHERS The usher is the platform responsible for catalysis of DSE and translocation of the multi-subunit pilus fibre across the outer membrane, while maintaining membrane integrity. It is an outer-membrane protein composed of five functional domains: a 24-stranded beta barrel channel, plug domain, N-terminal domain (NTD) and C-terminal domains (CTD1, CTD2) [77 79]. The usher is able to orchestrate the ordered assembly of multiple subunits into fibrous structures. Genetic studies combined with biophysical work on the kinetics of usher-catalysed reactions between usher domains and various subunits have provided novel models of macromolecular assembly.

Functions of the Usher Domains in Pilus Assembly Recent X-ray crystallography studies revealed that in the solitary usher (PapC), the plug domain resides in the lumen of the transmembrane channel, positioned to prevent the flow of molecules across the outer membrane [80,81]. The plug domain is thought to be kept in place by a β5-6 hairpin and an α-helix emerging from the main β-barrel porin structure; however, the details of the critical interactions keeping the plug in place remain unclear. When the usher is in complex with the chaperone adhesin, the plug domain is translocated into the periplasmic space, creating an unobstructed channel allowing the extrusion of assembled pilins [82]. Crystallography studies also revealed that the conformational change associated with the plug switch helps the β-barrel domain change conformations from the apo, kidney-shaped con˚ ) to the circular form (44 3 36 A ˚) formation (52 3 28 A [82]. This conformational change likely facilitates the ˚ in diameter) protrusion of folded pilins (B20 25 A across the OM as pilus biogenesis occurs. Following the movement of the plug domain towards the periplasmic space, it mediates a high-affinity, stable interaction with the permanently periplasmic N terminus (NTD) of the usher [82,83]. This high-affinity interaction likely stabilizes the usher protein in an open, active state. The plug domain and the two permanently periplasmic usher domains, NTD and CTDs, are responsible for the bulk of the assembly function of this molecular machine. Mutations in either NTD or CTDs or deletions of the plug domain completely inhibit pilus assembly, implicating their direct role in catalysis of pilus assembly [77,81,84,85]. Several biophysical and biochemical techniques have been used to better understand the role each usher domain has in assembly. Binding affinities of each usher domain for the chaperone subunit complexes were measured using purified proteins of P pilus components. NTD was shown to selectively bind the chaperone adhesin complex with the

highest affinity (KD 5 1 nM), while binding the minor tip chaperone subunit complexes with much lower affinity (KD 5 1 μM). Unlike NTD, plug and the plug NTD complex bound the chaperone and all chaperone subunit complexes with nearly equal affinity (KD 5 10 100 nM). Interestingly, CTD2 was shown to catalyse the dissociation of the tight complex that forms between the PapD PapG complex and the NTD of the PapC usher. CTD2 mediates the dissociation of the PapD PapG by binding to the chaperone adhesin, and not the NTD. In addition to binding the PapD PapG chaperone adhesin complex, CTD2 binds chaperone and all chaperone subunit complexes with equal affinity (KD 5 1 μM), but lacks affinity for the chaperone terminator complex (PapD PapH). The inability of the terminator to transfer to CTD2 may suggest that the plug and/or plug NTD bind this last subunit, terminating and anchoring the pilus. Overall, the high affinity that NTD has for the chaperone adhesin complex designates NTD as the initial recruitment site for the chaperone adhesin complex. Plug or the NTD plug complex recruits all other chaperone subunit complexes. CTD2, in addition to serving as a docking site, likely dissociates chaperone subunits from NTD and plug, with the exception of the chaperone terminator complex, which binds on plug or the NTD plug complex and terminates further pilus growth, while also helping to anchor the pilus on the bacterial cell surface (Fig. 8.2).

Usher Selectivity and Potential Binding Surfaces It is interesting that NTD can discriminate between subunits loaded onto the chaperone, despite the high structural homology that pilins share. A theory was proposed by Di Yu and colleagues to explain the significantly high affinity of chaperone adhesin complexes for NTD compared to chaperone alone using the Caf system of Yersinia pestis [86]. They identified a conserved chaperone diproline motif (residues P103-P104 of Caf1M) that goes through a conformational change upon subunit interaction. This mechanism, termed ‘proline lock’, was observed at the chaperone’s NTD targeting region, such that binding of the free chaperone to the pilin subunit caused an allosteric disengagement of the structural lock by rotating the proline lock away from the NTD targeting surface which it normally blocks. This conformational change was independent of classic proline cis trans isomerization. Mutagenesis and deletion studies of the region surrounding the proline lock caused a significant decrease in binding affinities of chaperone subunit complexes for the usher NTD [86]. Interestingly, subunitinduced changes on the chaperone may dictate the affinity of chaperone subunit complexes for NTD. It remains to

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be determined if the chaperone structural lock is a phenomenon that differentially influences NTD binding affinity depending on which subunit is bound to the chaperone. Another theory to explain these affinity differences is the differential hydrogen bonding capacities various chaperone subunit complexes display toward the NTD. Crystallography studies carried out on the type 1 pilus system indirectly suggest that some subunits (like FimG and FimA) cannot bind NTD because of an incomplete set of hydrogen bond interactions with the N-terminal tail of NTD that adopts an ordered conformation upon binding [73,78]. Unlike NTD, plug and CTD2 are involved in indiscriminate recruitment of all chaperone subunit complexes [83], with the exception in CTD2 for binding the chaperone terminator complex. CTD2 and plug share significant homology in terms of their topology, which may explain their non-selective recruitment functions. They both adopt a small β-sandwich fold where CTD2 has an extra β-strand at its C-terminus [87]. It remains to be evaluated if these domains use similar surfaces for chaperone subunit binding or exhibit unique recruitment mechanisms. In the Caf1A usher of the F1 capsule biogenesis system in Yersinia pestis, two conserved, solvent exposed hydrophobic patches located on CTD2 were shown to be required for capsule assembly. Double point mutations at the conserved sites significantly reduced F1 antigen assembly [88]. Statistical coupling analysis studies revealed that similar, co-evolving surface patches exist on CTD2 (Ford, Volkan & Hultgren, unpublished data). It remains to be seen if these residues are involved in the interaction of chaperone subunit complexes with CTD2 or if they may be involved in another as yet undetermined step during pilus assembly. Similarly to CTD2, the binding surface of the plug domain for chaperone subunit complexes remains unknown. A search of structural homologues revealed that the plug domain, which consists of an Ig-like fold, exhibits a significant three-dimensional homology to PapD (z-score: 3.4, rmsd: 2.6) (Ford, Volkan & Hultgren unpublished data) [89 91]. When the plug domain and the PapD chaperone were superimposed, it was clear that the plug is specifically homologous to the region of PapD that is directly involved in subunit binding and formation of PapD dimers [63]. It remains to be investigated whether the plug domain is binding to chaperone subunit complexes in a similar fashion as PapD binds to itself or to subunits.

Current Model of Chaperone Usher Pilus Biogenesis at the Usher The collective results and interpretations of the studies carried out so far suggest the following working model of CUP pilus assembly at the outer membrane (Fig. 8.2):

1. Initially, the usher adopts an inactive, gated conformation, in which the plug domain resides in the middle of the translocation pore. At this stage, the pore has a kidney-shaped structure while NTD and CTDs are likely disordered in the periplasmic space [80]. 2. Upon NTD’s high-affinity interaction with the initiator of pilus assembly, the chaperone adhesin complex, a conformational change in NTD likely occurs that triggers a conformational change in the plug to allow its protrusion into the periplasm. The binding energy of chaperone adhesin targeting to NTD may induce a movement in the NTD linker, which can help in the transition from ovular to circular conformation of the pore. This drastic change in pore structure may aid in translocation of the plug domain to the periplasmic space and/or prevent its re-entry into the channel lumen. 3. At this stage, the transfer of the chaperone adhesin from NTD to CTDs occurs through the catalytic dissociation function of CTD2. The NTD has a long flexible linker, so it may utilize this linker to approach the CTDs for cargo delivery. It remains to be investigated if concomitant movements of the NTD and CTDs may aid in this transfer. 4. The now free NTD can mediate its high-affinity, stable interaction with the plug domain and can recruit incoming chaperone subunit complexes while the chaperone adhesin complex is bound to the CTDs. An exclusive conformation in the NTD plug complex positions the tip subunit into the ideal orientation with respect to the adhesin for DSE to occur, allowing for the assembly of the growing pilus fibre. Upon successful subunit subunit interaction via DSE, the chaperone is displaced off the CTDs, leaving the CTDs available for the nascently incorporated chaperone subunit complex. This process repeats for all chaperone subunit complexes in the tip-to-base order observed in the mature pilus fibre. 5. The last step in assembly is the recruitment of the chaperone terminator by the NTD plug complex. Chaperone terminator complex of P pili, PapD PapH, was shown to be unable to bind CTD2 and unable to undergo DSE, as its P5 pocket is blocked. Hence, incorporation of the chaperone terminator complex to the pilus terminates pilus growth. With a stable interaction of the chaperone terminator complex with plug or the NTD plug complex, the mature fibre is anchored to the OM, ready to perform its adhesive function.

Structural Insights into the Growing Pilus The recent X-ray crystal structure of a fimbrial tip in complex with the chaperone and usher has provided new

154 PART | 1 Bacterial Structure

low-energy exit path while conformationally preparing the usher and the growing pilus for further chaperone subunit recruitment [92].

Pore-Gating Mechanisms of Usher

FIGURE 8.4 Snapshots of pilus assembly. (a) Crystal structure of the FimD C H complex (PDB ID 3RFZ). This provides a view of the first translocation event in process. The plug domain (blue cartoon) exits the FimD usher pore (orange cartoon) to bind the NTD (cyan cartoon), which allows FimH (green spheres) to protrude into the usher pore in an elongated conformation while bound to the FimC chaperone (yellow spheres), which itself is bound to CTD1 (magenta cartoon) and CTD2 (purple cartoon). (b) Crystal structure of FimD C F G H complex (PDB ID 4J3O). After a few steps of assembly, this structure reveals FimF (salmon spheres) bound to FimG (red spheres) bound to FimH (green cartoon) via donor strand exchange (DSE). FimF lies at the base of the assembled tip and binds FimC, which is again seen bound to CTD1 and CTD2.

insights regarding the structural basis for pilus assembly and growth [92]. The snapshot of this pilus biogenesis intermediate offers a view of the chaperone FimC mediating contacts between CTD of the FimD usher and FimF within the assembled FimF FimG FimH polymer, which passes through the usher pore (Fig. 8.4). At the distal end of this assembled tip, the adhesin FimH undergoes a significant structural change. While FimH adopts an elongated conformation prior to transport across the usher pore, the lectin domain swings closer to the pilin domain of FimH after its transport, likely to provide the necessary energy to facilitate in translocation of the growing fibre. It is thought that, as subsequent subunits of the pilus pass through the β-barrel, these structural changes in FimH prevent the pilus from retreating back through the pore towards the periplasmic space, as well as aid in efficient translocation [92]. The structural studies also revealed specific binding sites for the adhesin and the subunits, 180 apart within the usher pore lumen, helping the pilus to retain a central position within the pore as extrusion takes place. The anticlockwise rotational path that the pilus follows during extrusion into the extracellular milieu provides a

CUP ushers are among the largest single protein outer membrane β-barrel structures, composed of 24 β-strands. ˚ x 25 A ˚ in diameter and The PapC central channel is 45 A thus could potentially allow passage of molecules from the extracellular milieu, which could be detrimental to the bacterial cell. Hence, there is an inherent need for strict pore-gating mechanisms to retain OM impermeability and sustain bacterial homeostasis. Previous studies revealed that the plug domain, which resides in the middle of the translocation pore, gates the solitary usher pore. Interestingly, planar lipid bilayer electrophysiology studies carried out on PapC usher demonstrated that the wild-type usher pore is mostly closed with short-lived, frequent transitions to open states. Studies of the involvement of the β5-6 hairpin loop and the usher α-helix in pore gating have demonstrated that deletion of either caused the usher pore to remain significantly more open. As expected, removal of the plug domain resulted in a channel with extremely large conductance [85]. The impact that the NTD and CTDs may have on the pore-gating properties of the usher remains elusive. A structure function analysis carried out using antibiotic sensitivity revealed the mechanism by which the α-helix maintains the plug domain in the usher pore. A salt bridge interaction between plug (R305) and α-helix (E567) has been observed such that mutating either residue favours the usher pore in an open state, signifying the mechanism of α-helix involvement in pore gating (Volkan & Hultgren unpublished data). Flipping the residue locations regenerated electrostatic contact and reverted the antibiotic sensitivity phenotype to WT levels. Interestingly, despite a significant increase in the open conformation of the usher when the salt bridge is mutated, functional pilus biogenesis was not affected. This reveals the distinction between the pore gating and pilus assembly functions of the usher. It is likely that during pilus biogenesis, recruitment of the chaperone adhesin by the usher NTD triggers a conformational change that breaks the R305 E467 bond, resulting in the translocation of the plug to the periplasm and thus converting the usher to an open state. Overall, pilus assembly at the usher is achieved by cooperative functions of each of the five domains of the usher. These domains work in concert to recruit chaperone subunit complexes and assemble the pilus while strictly regulating pore permeability to retain bacterial homeostasis.

Chapter | 8 Pili and Fimbriae of Gram-Negative Bacteria 155

ALTERNATIVE CHAPERONE USHER PATHWAYS Composite, rigid structures exemplified by P and type 1 pili are not the only pilus types assembled by chaperoneusher pathways. The thinner, simpler, more flexible fimbriae structures of K88, K99 and P987 from ETEC and F1 antigen from Y. pestis are also assembled by CUP. These pili are generally involved in adhesive functions without an exclusive subunit dedicated to adhesion. Instead, the major pilus subunit, which gives rise to the entire pilus, is involved in adhesion [93]. For instance, the 2 4-nm-thick K88 fibres mediate adherence to intestinal cells [94], causing neonatal diarrhoea in some farm animals. Similarly, F1 antigen from Y. pestis, the causative agent of plague, is also assembled by CUP where F1 antigen fibres are solely composed of a single subunit, Caf1. Interestingly, Caf1M and Caf1A proteins, employed as the usher and the chaperone, respectively, share significant homology with the chaperone and the usher from the P pilus system, PapC and PapD [63,86]. While this homology suggests similarities in the assembly mechanisms of the two pilus systems, the Pap system assembles complex, bipartite, composite pili while the Caf system assembles flexible, fibrous, thinner structures, demonstrating the diverse features of CUP. Another variant of the classical CUP is the Coo system (CS1 family) expressed by ETEC. Similarly to K88 and F1 antigen, these fibres are predominantly composed of a single major pilus subunit termed CooA [95,96]. Components of the CS1 system share no detectable primary sequence similarity with those from the classical CUP system [97 99]. Only four linked genes, CooA, CooB, CooC and CooD, are sufficient in assembly of functional CS1 fibres [97 100]. CooC is thought to serve as the usher through which translocation of the subunits occurs [95]. CooA is the major pilin subunit, which forms the bulk of the pilus. Contrasting this system to that of F1 antigen and K88 is that there is another minor subunit, CooD, localized at the tip and required for adherence, likely acting as an adhesin [101]. Both CooA and CooD are stabilized by the CooB protein, which likely plays a role as the chaperone [51,95]. Interestingly, in addition to stabilizing the subunit, CooB is also involved in stabilization of the usher protein, which is a unique phenomenon not frequently observed in CUP systems. Since the assembly of CS1 pili depends on specialized periplasmic chaperones distinct from those of the classical CUP systems, this assembly mechanism is termed the ‘alternate chaperone pathway’.

DIVERSITY OF CUP SYSTEMS IN DISEASE CUP systems are extensively used by various bacterial species to mediate adhesion. For instance, enteric pathogens such as enterotoxigenic E. coli (ETEC) encode

numerous pilus systems that are needed for attachment to host surfaces. ETEC encode several colonization-factor antigen (CFA) pili that are necessary for colonization of the intestine. For instance, K99 pili encoded by ETEC are needed to mediate attachment to N-glycoyl neuraminic acid-GM3 on host small intestine such that bacteria can multiply and secrete toxins to cause diarrhoea in domestic animals like piglets and lambs [102 104]. K88 and 987P pili also encoded and expressed by ETEC are involved in colonization of the intestinal tract, as well [105 109]. Salmonella species including S. typhimurium express long polar fimbriae (Lpf) and plasmid-encoded fimbriae (Pef). Lpf is involved in colonization of murine intestinal mucosa [110 112], whereas Pef pili are involved in Salmonella adhesion to murine intestinal epithelium, which results in excessive fluid accumulation in the intestines [113].

DIFFERENT PILUS SYSTEMS INVOLVED IN ADHESION AND DISEASE Various types of pili other than CUP pili are used by Gram-negative pathogens to mediate disease. For instance, Vibrio cholerae pathogenesis depends on a dual function pilus, the toxin co-regulated pilus (TCP). TCP is needed for binding to small intestinal cell surfaces [114], where it is also used as a receptor for the lysogenic phage (CTX\ϕ) that encodes the two subunits of cholera toxin [8]. TCP is thought to be involved in interbacterial interactions, allowing the transfer of the encoded toxin within V. cholerae strains [115,116]. During the infection process, adhesive pili are conveniently situated between the host and the bacterial cell, such that a crosstalk between the two organisms can be initiated. Upon carrying out their adhesive function, pili can simultaneously impact signal transduction processes of host cells. For instance, the type IV pilus from pathogenic Neisseria spp. promotes bacterial adhesion to epithelial and endothelial cells by binding to complement regulatory protein CD46, which consequently triggers host cell signalling responses [117,118]. Signal transduction cascades that occur following type IV pilus-mediated adhesion induce local elongation of microvilli towards the bacteria, leading to internalization [119,120]. Furthermore, the Neisseria type IV pilus acts in concert with its porin to induce fluctuations in host cell cytosolic Ca21 levels, an important second messenger controlling a multitude of eukaryotic cellular responses [117,121]. Likewise, both P and type 1 pili are capable of inducing signal transduction pathways in host cells during urinary tract infections. Prior reports suggest that binding of type 1 piliated bacteria to murine bladder results in increased inflammatogenicity, which promotes neutrophil influx [122], and that P pilus binding

156 PART | 1 Bacterial Structure

TABLE 8.1 Adhesive Fibres from Gram-Negative Bacteria Fibre

Assembly Proteins

Adhesin

Organisms

Associated Diseases

P pili

PapD and PapC

PapG

Escherichia coli

Pyelonephritis

S pili

SfaE and SfaF

SfaS

Escherichia coli

Neonatal meningitis

Type 1 pili

FimC and FimD

FimH

Escherichia coli, Klebsiella pneumoniae, Salmonella spp.

Cystitis

F1 Antigen

Caf1A and Caf1M

Caf1

Yersinia pestis

Plague

Pef pili

PefD and PefC

Unknown

Salmonella enterica serovar Typhimurium (Salmonella typhimurium)

Gastroenteritis

Long polar fimbriae (Lpf)

LpfB and LpfC

Unknown

Salmonella typhimurium

Gastroenteritis

K99

FaeE/FaeD

K99 (major component)

Escherichia coli

Neonatal diarrhoea in piglets, calves, lambs

K88

FanE/FanD

K88 (major component)

Escherichia coli

Neonatal diarrhoea in piglets

987P

FasB/FasD

987P (major component)

Escherichia coli

Diarrhoea in piglets

MR/K (type 3) pili

MrkB and MrkC

MrkD

Klebsiella pneumoniae

Pneumonia

Type IV pili

PilQ, PilB and PilC

PilYI

Neisseria meningitidis

Meningitis

Hif pili

HifB and HifC

HifE

Haemophilus influenzae

Otitis media and meningitis

CS1 pili (Alternative Chaperone Pathway)

CooB and CooC

CooD

Escherichia coli

Diarrhoea

to uroepithelial tissue stimulates the release of second messenger compounds like ceramides [123,124]. These findings suggest that pili are not only involved in adherence but also use adhesion to modulate host responses and reaction to infection. A list of representative, extensively studied adhesive fibres from Gram-negative bacteria is detailed in Table 8.1.

CURLI: EXTRACELLULAR NUCLEATION/ PRECIPITATION PATHWAY Many members of the Enterobacteriaceae such as Escherichia, Salmonella and Citrobacter assemble thin adhesive fibres as part of their extracellular matrix [125,126]. These structures were sometimes referred to as thin aggregative pili in the 1980s until the late 1990s

[127,128]. This irregular class of highly aggregated structures is now most commonly referred to as ‘curli’. Curli, expressed by enteric bacterial species, share significant biochemical and structural similarities with diseasecausing eukaryotic protein fibres called amyloids, which are involved in degenerative diseases like Parkinson’s and Alzheimer’s [129]. The amyloid fibres expressed by Salmonella and Escherichia spp. are thought to mediate surface and cell cell contacts that promote biofilm formation and hence host colonization [19,125,130,131]. Curli can bind to the dye Congo red such that curliated bacteria growing on media supplemented with this dye stain red, providing a convenient tool to study genetic and biochemical aspects of curli biogenesis [132]. Curli genes are expressed optimally at 26 C from two divergently transcribed operons [133,134]. At least six genes are necessary for efficient curli expression and biogenesis: csgBA(C)

Chapter | 8 Pili and Fimbriae of Gram-Negative Bacteria 157

and csgGFED. The csgBA(C) operon encodes the structural subunits CsgA, which is secreted towards the extracellular milieu making the bulk of the fibre, and CsgB, which is thought to function as a fibre nucleator [134 137]. CsgA and CsgB mediate interactions on bacterial cell surface to give rise to the insoluble aggregative fibres [135,138]. CsgD activates the expression of these two proteins and is necessary for expression of the csgBA(C) operon. CsgG encodes the outer-membrane lipoprotein CsgG, which is conserved across the majority of known bacterial species and is involved in curli fibre assembly, nucleation and export [139 141]. Bioinformatic analyses helped reveal that CsgG utilizes α-helices to span the outer membrane of Gram-negative bacteria and form homooligomeric pores to facilitate subunit transfer and assembly [141,142]. Recent studies revealed that CsgG functions in cooperation with CsgE and CsgC. CsgC is encoded exclusively by a subset of Enterobacteriaceae such as Escherichia and Salmonella spp. [142,143]. A gene product for csgC was not detected for a long time and its role in biogenesis remained elusive [134,144]. However, recent structural work revealed CsgC to be a β-sandwich accessory protein with an Ig-like structure [142]. Interestingly, the CsgC N-terminus was shown to be related to the innermembrane protein DsbD and to carry an oxidoreductase function [142]. It has been hypothesized that CsgC may create or remove disulphide bonds that cross-link CsgG transmembrane helices, which may influence CsgG pore size or selectivity [142]. One of CsgC’s crucial roles appears to be regulating insertion or pore behaviour of the outer-membrane curli assembly protein CsgG, as deletion of CsgC interferes with curli-dependent biofilm formation [142]. CsgE is thought to be involved in regulating proper spatial and temporal assembly of CsgA subunits by prohibiting self-assembly of CsgA into amyloid fibres, ensuring that CsgA assembly solely occurs on the cell surface [143]. Simultaneously, CsgE provides substrate specificity to the outer-membrane pore CsgG, preventing premature CsgA assembly. Thus, CsgE and CsgG likely work together to keep the bacterial cell safe from the toxic effects of intracellular amyloidogenesis. Similarly, housekeeping and stress-induced chaperones like DnaK, Hsp33 and Spy are thought to be involved in prevention of premature CsgA interactions during curli biogenesis [145]. CsgF is another curli assembly protein, which interacts with CsgG and regulates the secretion of curli subunits in an assembled fashion to the extracellular milieu [140,141,146]. In the absence of CsgF, curli subunits are released to the media without efficient polymerization [146]. This cell surface-associated protein is necessary for CsgB (nucleator) localization and is critical for nucleation of amyloid subunits into fibres [146].

INTERFERING WITH THE PILUS Currently, there is an increasing need for novel innovations in antimicrobial therapy, as rising antibiotic resistance and an epidemic of antibiotic-resistant strains pose challenges in the treatment of microbial diseases [147,148]. A further complicating issue is that, despite similarity between genomic compositions of different strains of E. coli, for instance, their virulence profiles tend to differ significantly, seemingly due to differing gene expression levels, interplay between genes, and/or genetic variations [149,150]. A riskier issue is that there has been an accelerated rate of global transmission of multi-drug-resistant pathogens. For instance, some uropathogens are becoming increasingly resistant to not only the first-line antibiotics like trimethoprim-sulphamethoxazole (TMP-SMX) but also to second-line therapies like fluoroquinolones [151 153]. In addition to the well-acknowledged problem of antimicrobial resistance in the clinical setting, particularly in the past 70 years, there has been an enrichment of antibiotic resistance genes in the environmental microbiota like soil bacteria due to excessive antibiotic use [154]. The controversial large-scale usage of conventional antibiotics in farming industries can also influence emergence and spread of multi-drug-resistant strains [155 157]. A new approach in fighting microbial disease is to inhibit virulence-associated functions of bacteria, as opposed to bacterial killing or slowing bacterial growth, thereby avoiding damage to host microbiota while decreasing chances for the evolution of bacterial resistance mechanisms. By inhibiting virulence factors, the pathogen is deprived of its ability to persist, form biofilms and evade innate defences, thus resulting in clearance by the host (reviewed in [158]). CUP pili are virulent appendages involved in multiple diseases ranging from UTIs [26,27,159] to neonatal meningitis [30], making them attractive targets for antivirulence therapeutics. Studies carried out on type 1 pili, which are required for cystitis, revealed promising results using anti-virulence compounds for UTI treatment and prevention [160]. The adhesin of type 1 pili, FimH, binds to mannosylated uroplakin of uroepithelial cells, facilitating the internalization of UPEC, which go on to form biofilm-like intracellular bacterial communities (IBCs) [25,28]. Taking advantage of this knowledge, alkyl- and phenyl-α-mannopyranosides (mannosides) were designed to competitively inhibit binding of FimH to host uroplakin. Mannosides were shown to not only block adhesion but also prevent internalization of bacteria and IBC formation in an established murine model [160]. Furthermore, mannosides potentiated the efficacy of existing antibiotics in addition to inhibiting biofilm formation on biotic/abiotic surfaces such as catheters [160,161].

158 PART | 1 Bacterial Structure

In addition to interfering with pilus function by targeting the adhesin, efforts in preventing de novo pilus biogenesis have also been successful. Rationally designed bicyclic 2-pyridone compounds (pilicides) were designed to bind the CUP chaperone. X-ray crystallography has confirmed that pilicides bind the P pilus chaperone, PapD, on the usher-binding site on the N-terminal domain (see section entitled ‘Conserved Chaperone Surfaces and Donor Strand Complementation’). This chaperone pilicide interaction was shown to inhibit binding of chaperone subunit complexes to the usher by surface plasmon resonance experiments. Moreover, pilicides abolished P and type 1 pilus biogenesis as determined by haemagglutination assays [74]. In fact, the pilicide-binding site lies near a recently uncovered ‘proline lock’ site, which undergoes a conformational change upon subunit binding (see section entitled ‘Usher Selectivity and Potential Binding Surfaces’) [86]. The pilicides may bind and trigger a conformational change in this site, thereby impacting subunit binding or causing conformational changes in the chaperone, driving chaperone subunit separation. Alternatively, the pilicide may simply act by sterically inhibiting chaperone subunit complexes from binding to the NTD of the usher. To meet the important challenge of developing novel antimicrobial therapeutics, mannosides and pilicides provide an interesting, innovative mode of slowing down bacterial resistance while leaving host microbiota and homeostasis untouched. Translated to clinical practice, pilicides and mannosides may one day be useful for disease prevention, treatment or as adjuvants to increase antibiotic efficacies [160]. In an era of bacterial resistance when there is a vital need for novel approaches, anti-virulence therapeutics pose an exciting opportunity for fighting microbial disease while steering clear from antimicrobial resistance.

[7]

[8]

[9] [10]

[11] [12] [13]

[14]

[15] [16] [17]

[18]

[19]

[20]

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