Summary The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two‐hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N‐terminal motif in PilN is essential for both PilM–PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

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,...

Pili (or fimbriae) are hair-like appendages that extend from the surface of many bacteria, and are polymers of primarily one protein generically named pilin. Out of the many types of pili that have been identified and classified according to their morphological and/or molecular characteristics, type IV pili (Tfp) are undoubtedly the most widespread. This chapter describes the biosynthesis, morphological, molecular characteristics, and functions of Tfp.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2′O-Methyl (2′OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2′OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2′OMe AON and LNA/2′OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2′OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

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