Motor-independent retraction of type IV pili is governed by 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 likely diminish pilin–pilin interactions within the filament to produce less-stable pili. One mutation adds a bulky residue to α1C, a universally conserved feature of T4P. We found that inserting a bulky residue into α1C of the retraction motor–dependent Acinetobacter baylyi competence T4P enhances motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor–independent, V. cholerae toxin-coregulated T4P stabilizes the filament and diminishes pilus retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor–independent T4P, gram-positive 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, in part, on the inherent instability of the pilus filament, which may be a conserved feature of diverse T4Fs. This provides evidence for a long-standing yet previously untested model in which pili retract in the absence of a motor by spontaneous depolymerization.

The PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus inVibrio cholerae

“…Some T4aP pili can undergo motor-independent retraction via the spontaneous depolymerization of the pilus filament [41]. Taking this into account, there are at least two distinct mechanisms by which PilT could support MSHA biogenesis.…”

“…One possibility is that PilT actually drives processive pilus extension via its ATPase activity. It has been shown in a number of T4P systems that the ATPase activity of the motor that drives dynamic activity correlates with the speed of extension/retraction [15, 41, 43, 44]. Thus, if PilT’s ATPase activity drives processive extension, we hypothesized that a mutation that slows its ATPase activity should correspondingly slow down extension speed.…”

“…Thus, if PilT’s ATPase activity drives processive extension, we hypothesized that a mutation that slows its ATPase activity should correspondingly slow down extension speed. Previous work has defined mutant alleles of T4P motor ATPases that reduce ATPase activity ~1.5-2-fold [15, 41, 43, 45]. Therefore, we generated mutants of both pilT ( pilT L201C ; aka pilT slow ) and mshE ( mshE L390C ; aka mshE slow ) to slow their ATPase activity, and we tested the impact of these mutations on MSHA extension speed.…”

“…This is supported by the observation that the MshA P31S suppressor allele can promote assembly of the MshA parent major pilin into a pilus filament. Additionally, we have recently shown that the packing of major pilin subunits within the pilus filament plays an important role in regulating motor-independent retraction of T4aP [44]. This suggests that the major pilin and the structure of the pilus filament are factors that likely contribute to the regulation of both T4aP extension and retraction.…”

“…Together, these data suggest that in addition to its defined role in promoting MSHA retraction, PilT also counterintuitively participates in the biogenesis of MSHA pili. Some T4aP pili can undergo motor-independent retraction via the spontaneous depolymerization of the pilus filament [44]. Therefore, there are at least two distinct mechanisms by which PilT could support MSHA biogenesis.…”