Type IV pili share a conserved mechanism of motor-independent retraction that is an inherent property of the pilus filament

Abstract

Type IV pili (T4P) are dynamic surface appendages that promote virulence, biofilm formation, horizontal gene transfer, and motility in diverse bacterial species. Pilus dynamic activity is best characterized in T4P that use distinct ATPase motors for pilus extension and retraction. Many T4P systems, however, lack a dedicated retraction motor and the mechanism underlying this motor-independent retraction remains a mystery. Using the Vibrio cholerae competence pilus as a model system, we identify mutations in the major pilin gene that enhance motor-independent retraction. These mutants produced less stable pili, likely due to diminished pilin-pilin interactions within the filament. One mutation adds a bulky residue to α1C, a universally conserved feature of type IV pilins. We found that inserting a bulky residue into α1C of the retraction motor-dependent Acinetobacter baylyi competence T4P is sufficient to induce motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor-independent V. cholerae toxin-co-regulated T4P stabilizes the filament and prevents retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor-independent T4P, Com pili, and type II secretion systems generally encode larger residues within α1C oriented toward the pilus core compared to retraction motor-dependent T4P. Together, our data demonstrate that motor-independent retraction relies on the inherent instability of the pilus filament that may be conserved in diverse T4Fs. This provides the first evidence for a long-standing, yet untested, model in which pili retract in the absence of a motor by spontaneous depolymerization.

Fresh extension ofVibrio choleraecompetence type IV pili predisposes them for motor-independent retraction

“…So far, our data show that properties that are inherent to the pilus fiber are important for regulating motor independent retraction. Although our data demonstrate that PilB is not required for motor-independent retraction via its ATPase activity (25) or via its presence to align the T4P machine (Fig. 2), other T4P components may play an important role in regulating this spontaneous depolymerization (e.g.…”

“…6) remains lacking. This model is largely bolstered by a previous suppressor screen where the only mutations that could enhance motor-independent retraction all mapped to the pilA gene (25). These mutations disrupted the stability of the pilus fiber to enhance motor-independent retraction, which suggested that the mechanism underlying retraction was an inherent property of the pilus fiber.…”

“…A simple explanation for this would be that PilB is a bifunctional motor powering both extension and retraction in the absence of PilT and PilU (24). However, our recent findings suggest that PilB is not a bifunctional motor because reducing the ATPase activity of PilB did not affect motorindependent retraction rates (25). Although PilB does not power ΔpilTU retraction via its ATPase activity, it remains possible that it regulates this process.…”

“…All T4P systems encode a predicted extension ATPase, commonly called PilB, that facilitates pilus polymerization (20,21). Canonically, pilus retraction is mediated by a dedicated retraction ATPase (22,23), however two other modes of pilus retraction have also been described: a bifunctional ATPase that promotes both pilus extension and retraction (24); and pilus retraction that occurs without the aid of any ATPase motors (12,25,26). Many T4P systems likely rely on motor-independent retraction, and even many motor-dependent T4P have recently been shown to retract in the absence of their dedicated motors (10,11,26).…”

“…The extension ATPase PilB and the retraction ATPases PilT and PilU drive the dynamic assembly and disassembly of competence pili, respectively. Previous work shows that PilB is required for pilus dynamic activity and that a ΔpilB mutant does not produce pili (4,25,32). This system has recently been used to study motor-independent retraction because a mutant strain lacking both retraction motors (ΔpilTU) is capable of motor-independent retraction at a reduced frequency (10,22,25).…”