Pseudomonas aeruginosa Minor Pilins Are Incorporated into Type IV Pili

Type IV pili are long filamentous appendages required for both adhesion and a unique form of motility known as twitching. Twitching motility involves the extension and retraction of the pilus and requires a number of gene products, including five conserved pilin-like proteins of unknown function (FimU, PilV, PilW, PilX, and PilE in Pseudomonas aeruginosa), termed ‘minor’ pilins. Maintenance of a specific stoichiometric ratio among the minor pilins was important for function, as loss or overexpression of any component impaired motility. Disruption of individual minor pilin genes, or of the AlgR positive regulator of minor pilin operon expression in a strain where pilus retraction was blocked by inactivation of the PilT retraction ATPase, revealed that pili were produced, although levels of piliation were reduced relative to pilT positive control. Differences in the levels of piliation of complemented strains pointed to specific roles for each protein in the assembly process, with FimU and PilX being implicated as key promoters of pilus assembly on the cell surface. Using specific antibodies for each protein, we showed that the minor pilins FimU, PilV, PilW, PilX, and PilE were processed by the pre-pilin peptidase PilD and incorporated throughout the growing pilus filament. This is the first study to demonstrate that the minor pilins, conserved among bacteria expressing type IVa pili, are incorporated into the fiber and support a role for them in the initiation, but not termination, of pilus assembly.     

Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.     

Structural Characterization of Novel Pseudomonas aeruginosa Type IV Pilins

Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-Å X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal α-helix and four-stranded antiparallel β-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the αβ-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA_PA14, compensatory changes allow for maintenance of a similar shape.