T4P Assembly Research Articles

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.

PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA

Pseudomonas aeruginosa type IV pili (T4P) are virulence factors that promote infection of cystic fibrosis and immunosuppressed patients. As the absence of T4P impairs colonization, they are attractive targets for the development of novel therapeutics. Genes in the pilMNOPQ operon are important for both T4P assembly and a form of bacterial movement, called twitching motility, that is required for pathogenicity. The type II membrane proteins, PilN and PilO, dimerize via their periplasmic domains and anchor this complex in the inner membrane. Our earlier work showed that PilNO binds PilP, a periplasmic lipoprotein (S. Tammam, L. M. Sampaleanu, J. Koo, P. Sundaram, M. Ayers, P. A. Chong, J. D. Forman-Kay, L. L. Burrows, and P. L. Howell, Mol. Microbiol. 82:1496-1514, 2011). Here, we show that PilP interacts with the N0 segment of the outer membrane secretin PilQ via its C-terminal domain, and that the N-terminal cytoplasmic tail of PilN binds to the actin-like protein PilM, thereby connecting all cellular compartments via the PilMNOPQ protein interaction network. We show that PilA, the major pilin subunit, interacts with PilNOPQ. The results allow us to propose a model whereby PilA makes extensive contacts with the transenvelope complex, possibly to increase local concentrations of PilA monomers for polymerization. The PilNOP complex could provide a stable anchor in the inner membrane, while the PilMNOPQ transenvelope complex facilitates transit of the pilus through the periplasm and clamps the pilus in the cell envelope. The PilMN interaction is proposed to be responsible for communicating signals from the cytoplasmic to periplasmic components of this complex macromolecular machine.

Structure of an Essential Type IV Pilus Biogenesis Protein Provides Insights into Pilus and Type II Secretion Systems

Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.

Ultrahigh Resolution and Full-length Pilin Structures with Insights for Filament Assembly, Pathogenic Functions, and Vaccine Potential

Pilin proteins assemble into Type IV pili (T4P), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. However, challenges in crystallizing full-length fiber-forming and membrane protein pilins leave unanswered questions regarding pilin structures, assembly, functions, and vaccine potential. Here we report pilin structures of full-length DnFimA from the sheep pathogen Dichelobacter nodosus and FtPilE from the human pathogen Francisella tularensis at 2.3 and 1 Å resolution, respectively. The DnFimA structure reveals an extended kinked N-terminal α-helix, an unusual centrally located disulfide, conserved subdomains, and assembled epitopes informing serogroup vaccines. An interaction between the conserved Glu-5 carboxyl oxygen and the N-terminal amine of an adjacent subunit in the crystallographic dimer is consistent with the hypothesis of a salt bridge between these groups driving T4P assembly. The FtPilE structure identifies an authentic Type IV pilin and provides a framework for understanding the role of T4P in F. tularensis virulence. Combined results define a unified pilin architecture, specialized subdomain roles in pilus assembly and function, and potential therapeutic targets.

Weapons of mass retraction

Twitching motility is a unique form of bacterial propulsion on solid surfaces associated with cycles of extension, tethering and retraction of type IV pili (T4P). Although investigations over the last two decades in a number of species have identified the majority of the genes involved in this process, we are still learning how these pili are assembled and the mechanics by which bacteria use T4P to drag themselves from one place to another. Among the puzzles that remain to be solved is the mechanism by which hydrolysis of ATP is coupled to pilus assembly and disassembly, and how the cell envelope structure is modified to accommodate the passage of the pilus through the periplasm. Unravelling of these and other enigmas in the T4P system will not only teach us more about these important colonization and virulence factors, but also help us to understand related processes such as type II secretion, which relies on a set of proteins homologous to those in the T4P system, and bacterial conjugation, involving retractable pili belonging to the F-like subgroup of the type IV secretion family. This review focuses on recent discoveries relating to the assembly and function of T4P in generation of twitching motility.