Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

Abstract

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gramnegative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.type IV pili | type IV filamentous nanomachines | filament assembly | synthetic biology E volution has provided prokaryotes with sophisticated surface nanomachines that endow them with many functions instrumental to their ability to colonize most niches on Earth. Among these engineering marvels, type IV filamentous (Tff) nanomachines (1), of which type IV pili (Tfp) are the paradigm, are unique for two reasons. They are exceptionally (i) widespread, with genes encoding distinctive proteins found in virtually every prokaryotic genome, and (ii) multipurpose, associated with functions as diverse as adhesion, motility, protein secretion, DNA uptake, electric conductance, and so forth (1). Much of this broad distribution and multifunctionality is due to Tfp (1).All Tff nanomachines share multiple components and are thought to use common basic operating principles. They have at their core a filament, which can be long or short and is a polymeric assembly of a protein named pilin, PilE in our model Tfp-expressing species Neisseria meningitidis (meningococcal nomenclature will be used here). Type IV pilins are produced as prepilins with a distinctive N-terminal class III signal peptide (2), consisting of a short hydrophilic leader peptide followed by a stretch of 21 hydrophobic residues, always forming an extended α-helix (3). This signal peptide is first recognized by the Sec machinery (4, 5), which translocates prepilins across the cytoplasmic membrane, where they remain embedded as bitopic proteins. The leader peptide is then cleaved by an integral membrane aspartic protease (6, 7), the prepilin peptidase PilD. This processing, which does not require other Pil proteins (8), is a prerequisite for polymerization of pilins into filaments. Filaments are helical polymers in which the pilins' extended N-terminal α-helices are buried within the filament core, almost parallel to its long axis (9). Finally, in gram-negative Tfp-expressing bacteria, filaments cross the outer membrane through a pore formed by the secretin PilQ (10).The molecular mechanisms of filament assembly remain poorly understood. However,...

The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

“…In this context, it is important to notice that interaction between traffic ATPases and PilM orthologs is not consistent throughout all T4P systems. For the traffic ATPase PilF in N. meningitidis, no interaction between the two proteins was detectable in bacterial two-hybrid assays [36], whereas for the T4P traffic ATPases PilB of M. xanthus and of P. aeruginosa and for heterologously produced PilF of N. meningitidis, direct interaction with PilM has been shown [33,42,43]. In the first two systems, PilB was also shown to interact with the integral membrane protein PilC [33,49].…”

“…The conserved PilM, PilN, and PilO proteins of different T4P systems have been shown to interact with each other [21, [36][37][38][39][40][41][42]. Moreover, PilM of the T4P systems of M. xanthus, Pseudomonas aeruginosa, and Neisseria meningitidis were found to interact with the traffic ATPase PilB/PilF [33,42,43]. Studies of T4Prelated T2SS, such as the T2SS of V. cholerae, Erwinia chrysanthemi, and Xanthomonas campestris, also revealed interactions between traffic ATPases and PilM homologs [30,44,45].…”

Calcium transcriptionally regulates movement, recombination and other functions of Xylella fastidiosa under constant flow inside microfluidic chambers

“…Genes encoding PilN, PilO and PilP for TFP pili assembly subcomplex were upregulated. These proteins, together with PilQ and PilM, constitute the secretion complex of the pilin protein that is the basic unit for the extracellular part of TFP (Goosens et al , ). Mutation of this complex leads to absence of TFP (Li et al , ).…”

Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli

“…We show that, in addition to PpdD and a prepilin peptidase, HofC and four minor pilins together are essential for pilus assembly on the bacterial surface. The essential role of the HofC homologue in P. aeruginosa PilC (Takhar et al , ) has been recently confirmed for N. meningitidis PilG (Goosens et al , ). In the latter study, a synthetic approach was employed to assemble meningococcal T4P in the periplasm of E. coli and only eight proteins were essential.…”