Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction

Abstract

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. IMPORTANCE Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

Pseudomonas aeruginosa distinguishes surfaces by stiffness using retraction of type IV pili

“…6 ), TFP also exhibited a force-induced reversible stretching under extensile loads likely due to a conformational change in the PilA monomers. This unfolding-like feature was first demonstrated in gonococcal TFP and has also been shown for TFP in Vibrio cholerae and P. aeruginosa ( 44 – 48 ). As shown in SI Appendix , Fig.…”

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

“…If PilT is needed to maintain extended MSHA pili in this manner, we hypothesized that depleting PilT protein from piliated cells should result in the loss of surface pili due to the subsequent motor-independent retraction of those pili. The depletion of PilT was accomplished by adapting a previously described orthogonal degron system from Mesoplasma florum [42,43]. The translational fusion of a degron tag (called pdt2) to the C-terminus of native PilT (i.e., PilT-pdt2) allows for the specific and temporally regulated degradation of PilT by induction of the cognate M. florum Lon protease (mf-Lon).…”

“…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.…”

Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus

“…As a control, we also ectopically expressed V. cholerae pilT ( pilT Vc ). Competence pili in V. cholerae promote DNA uptake during natural transformation, a function that requires both pilus extension and retraction (8, 14). Thus, we functionally assessed competence pilus dynamic activity by performing natural transformation assays.…”

“…Thus, we opted to bypass the natural mechanism of competence induction in V. cholerae by ectopically expressing the master competence regulator TfoX ( P tac -tfoX ) and inactivating the quorum sensing negative regulator LuxO (Δ luxO ) as previously described (8). Chitin-independent natural transformation assays were performed exactly as previously described (8, 14). Briefly, strains were grown overnight in 3 mL of LB broth containing 100 μM IPTG at 30 °C rolling.…”