Tad Pili Play a Dynamic Role in Caulobacter crescentus Surface Colonization

Abstract

Bacterial surface attachment is mediated by filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions to mimic natural environments, we demonstrated that Tad pili undergo repeated dynamic cycles of extension and retraction. Within seconds after establishing surface contact, pilus retraction reorients cells into an upright position, promoting walking-like movements against the medium flow. Pilus-mediated positioning of the flagellate pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pilus dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells toward permanent surface attachment. IMPORTANCE Bacteria are able to colonize surfaces in environmental, industrial, and medical settings, where they form resilient communities called biofilms. In order to control bacterial surface colonization, microbiologists need to gain a detailed understanding of the processes that bacteria use to live at the liquid-surface interface and that allow them to adhere to and move on surfaces and eventually grow and persist on solid media. To facilitate these processes, bacteria are equipped with adhesive structures such as flagella and pili and with matrix components such as exopolysaccharides. How these cellular organelles are coordinated to optimize surface processes is currently subject to intense investigations. Here we used the model organism Caulobacter crescentus to demonstrate that polar pili are highly dynamic structures that are functionally interconnected with the flagellar motor to mediate surface sensing, thereby enforcing rapid and permanent surface attachment. These studies provide an entry point for an in-depth molecular analysis of bacterial surface colonization.

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae

“…Therefore, c-di-GMP-mediated maintenance of proper MSHA extension/retraction dynamics is likely key for mediating long-term V. cholerae surface attachment and downstream biofilm formation. A similar mechanism has been proposed in C. crescentus, where heightened c-di-GMP promotes Tad pilus retraction that not only aids in initial surface attachment, but also promotes flagellum-surface contacts that initiate production of the holdfast to facilitate long-term attachment 23 . However, even though correlations between secondary messenger signaling and pilus extension/retraction dynamics have been observed, the relationship between these signals/events are likely to be complex with both short-and long-term multigenerational temporal correlations.…”

“…Yet in other bacteria, c-di-GMP impact on type IV pilus production is mediated by different mechanisms. In Caulobacter crescentus, low to intermediate levels of c-di-GMP promote Tad pilus production, while high concentrations promote pilus retraction 23 . In Pseudomonas aeruginosa, while the PilB extension ATPase does not directly bind c-di-GMP 48 , a secondary protein FimX binds c-di-GMP to promote PilB localization to the assembly machinery and pilus elaboration [48][49][50][51][52] .…”

“…The c-di-GMP-binding motif observed in MshE is ubiquitous among many ATPases associated with type-II secretion system and type-IV pilus biogenesis 18,30,31 . While c-di-GMP is broadly conserved and is central to biofilm regulation among diverse bacterial species, our understandings of the direct molecular mechanism(s) by which c-di-GMP promotes type IV pilus production are still developing 13,23,27,28 . Here, we observe promotion of MshE extension activity at elevated c-di-GMP concentrations, resulting in a reduction of PilT-mediated retraction.…”

“…Many systems also contain a secondary depolymerization/retraction ATPase, which functions to retract the pilus from the cell surface 21 . Cycles of pilus extension and retraction have been shown to facilitate surface attachment, as well as surface-associated twitching motility within many bacterial species [22][23][24][25][26] . Despite the vital role and importance of the MSHA pilus in V. cholerae surface attachment, the dynamics of MSHA extension and retraction and their consequence on biofilm formation remain to be elucidated.…”

The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus

“…One possibility is that, upon surface contact, force generation leads to structural rearrangements within the filament that locks pili in the retraction state, which, in turn, prevents pilus extension and reincorporation of subunits, leading to elevated PilA levels in the inner membrane and subsequent activation of c-di-GMP signaling. Recent work showed that the second-messenger c-di-GMP itself also affects pilus dynamics, promoting increased activity at intermediate levels and retraction of pili at peak concentrations (49). Thus, blocking retraction of an individual pilus likely induces a positive feedback loop to induce retraction of additional pili at the cell pole, leading to robust surface sensing.…”