Mea­sur­ing Strength and Speed of the Mi­cro­bial Grap­pling Hook

by Am­ber Pol­lack-Berti

I'll ad­mit, I've been in love with the type IV pili (T4P) for a long time. Af­ter mem­o­riz­ing all those com­plex path­ways for reg­u­la­tion and me­tab­o­lism, there was some­thing so re­fresh­ing and ac­ces­si­ble about pili. These bac­te­r­ial sur­face ap­pendages are, by their na­ture, me­chan­i­cal struc­tures. They are easy to vi­su­al­ize. Their com­po­si­tion is sim­ple: a Type IV pilus is a flex­i­ble fil­a­ment made up of thou­sands of re­peat­ing pilin sub­units. Bac­te­ria em­ploy this one struc­ture for a va­ri­ety of pur­poses: T4P play struc­tural roles in motil­ity and biofilm for­ma­tion, and have also been im­pli­cated in DNA bind­ing and up­take, in host cell sig­nal­ing, and even in com­mu­ni­ca­tion of elec­trical sig­nals as 'nanowires'.

Hook. Source

T4P are long and thin and have great ten­sile strength. They are in­volved in bac­te­r­ial motil­ity us­ing a spe­cial mech­a­nism: the pili are re­tracted into the cell when the pilin sub­units are re­moved at the pilus' cell end by an AT­Pase, the pro­tein PilT. This re­sults in the ratch­et­ing of the pilus back into the cell.

Ex­per­i­men­tal setup and force gen­er­a­tion dur­ing T4P re­trac­tion. a) A cell is immo­bi­lized on a poly­styrene-coated cover slide. When a T4P bound to a bead in the laser trap is re­tracted, it dis­places the bead by a dis­tance d from the cen­ter of the trap. The force feed­back is trig­gered by mov­ing the slide by a dis­tance x to main­tain d at a con­stant value. b) Typ­i­cal de­flec­tion, d, of the bead dur­ing pilus re­trac­tion as a func­tion of time. F, force; t, time. Source

When a pilus is at­tached by its dis­tal end to a sur­face of agar, the re­trac­tion is so strong that the cell is pulled for­ward in a kind of move­ment called twitch­ing motil­ity. When a bac­terium at­taches via the pilus to the sur­face of a host cell, it re­tracts un­til the two come in con­tact. In these cases, the pilus acts as a grap­pling hook, pulling the cell along in short bursts of pili elon­ga­tion, attach­ment, and re­trac­tion.

The force ex­erted in pilus re­trac­tion as well as its speed have been re­cently mea­sured. Just how much force can be ex­erted dur­ing the re­trac­tion of one pilus? How fast can it re­tract? If you ap­ply force to the pilus, does it change the speed of re­trac­tion? These are physics ex­periments, al­beit at a mi­cro­scopic scale. The au­thors used laser tweez­ers to mea­sure the force and ve­loc­ity of pilus re­trac­tion. The ex­per­i­men­tal setup con­sists of im­mobilizing a cell on a mi­cro­scope slide and link­ing a bead to the end of a pilus. The bead can be lo­cal­ized via a laser trap, and the dis­tance it trav­els recorded as a func­tion of force and time.

Us­ing this ap­proach, they pit­ted two dif­fer­ent mi­crobes, Myx­o­coc­cus xan­thus and Neis­se­ria gonor­rhoeae, against one an­other in the equiv­a­lent of a bac­te­r­ial trac­tor pull. They com­pared M. xan­thus T4P to pre­vi­ously re­ported stats for N. gon­or­rhoeae. Could two dis­tinct bac­te­ria, liv­ing very dif­fer­ent lifestyles and gaz­ing at each other from far across the phy­lo­ge­netic tree, pull their pili with sim­i­lar force? Sim­i­lar ve­loc­ity?

Model of type IV pili as­sem­bly and re­trac­tion. Prepilin leader se­quences are cleav­ed by PilD. Processed PilA is as­sem­bled on a base of mi­nor pilins (PilE, V, W, X, and FimU) by the ac­tion of the cytoplas­mic mem­brane pro­tein PilC and the NTP-bind­ing pro­tein PilB. The pilus is extrud­ed through the outer mem­brane via a pore of PilQ mul­ti­mers. Pilus re­trac­tion is by the ac­tion of PilT. Source

When it comes to stalling force (the force at which re­traction drops by an or­der of mag­ni­tude), M. xan­thus may be the win­ner, with a stalling force of ~150 pN. But N. gon­or­rhoeae is no slouch, with a pre­vi­ously re­ported force of ~110 pN. What­ever the de­tails, the au­thors sta­te:"to our knowl­edge, the pilus mo­tor is the strongest lin­ear mo­tor re­ported in the lit­er­a­ture to date." By com­parison, the stalling force of an actin fil­a­ment is 7.7 pN per fil­a­ment, that of myosin 2.5 pN. But while both N. gon­or­rhoeae and M. xan­thus may re­tract their pili with the same gen­eral amount of force and at sim­i­lar velo­cities, there was a strik­ing dif­fer­ence: when force is ap­plied to N. gon­or­rhoeae there is an in­creased like­li­hood of di­rec­tion switch­ing: the pilus stops re­tract­ing and be­gins to elon­gate. In con­trast, ap­ply­ing force to a M. xan­thus pilus does not in­crease the like­li­hood of elon­ga­tion.

Sim­i­lar val­ues for re­trac­tion strength and ve­loc­ity sug­gest com­mon ma­chin­ery at work in two very dif­fer­ent bacte­ria. Does the dif­fer­ence in di­rec­tion switch­ing re­flect the dif­fer­ent lifestyles of a soil-dwelling mi­crobe and a hu­man pathogen? T4P sys­tems are em­ployed by a wide va­riety of bac­te­r­ial species. Cer­tainly stud­ies that investi­gate this mech­a­nism in other twitch­ing bac­te­ria will fur­ther ex­pand our knowl­edge of T4P retract­ion across di­verse bac­te­r­ial lifestyles.

Ref­er­ence

Clausen M, Jakovl­je­vic V, Sø­gaard-An­der­sen L, & Maier B (2009). High-force gen­er­a­tion is a con­served prop­erty of type IV pilus sys­tems. Jour­nal of Bac­te­ri­ol­ogy, 191 (14), 4633–4638. PMID 19429611

Am­ber is a Grad­u­ate Re­search As­sis­tant in the Ruby Lab at the Uni­ver­sity of Wis­con­sin, Madi­son, and the host of a blog that also fea­tures The Small Things: Tiny Top­ics (in­ac­tive since 2020).