Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements

Talà, Lorenzo; Fineberg, Adam; Kukura, Philipp; Persat, Alexandre

doi:10.1038/s41564-019-0378-9

Cited by 132 publications

(171 citation statements)

References 34 publications

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“…Previous reports have shown that PilU is necessary for twitching motility in P. aeruginosa [13], an activity that likely requires forceful retraction [30]. Consistent with this, our results indicate that PilU is necessary for maximal retraction force for the competence pilus in V. cholerae .…”

Section: Discussionsupporting

confidence: 91%

“…However, PilU is present in many type IVa pilus systems, so the question remains: Why is PilU so widely conserved if it is not necessary for pilus retraction? Recent work in Pseudomonas aeruginosa indicates that PilU may be required for forceful retraction [30]. Our data thus far indicate that pilus retraction in the pilU mutant is largely indistinguishable from the parent ( Fig 1B ), however, these measurements are based on free pili that are not retracting under load.…”

Section: Resultsmentioning

confidence: 53%

See 1 more Smart Citation

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Chlebek

Hughes

Ratkiewicz

et al. 2019

Preprint

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19Bacterial type IV pili are critical for diverse biological processes including horizontal gene 20 transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic 21 appendages extend and retract from the cell surface. In many type IVa pilus systems, extension 22 occurs through the action of an extension ATPase, often called PilB, while optimal retraction 23 requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog 24 of PilT called PilU. However, the function of this protein has remained unclear because pilU 25 mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is 26 relatively understudied compared to PilT. Here, we study the type IVa competence pilus of 27Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity 28 of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, 29 however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a 30 bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also 31 found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is 32 important for generating maximal retraction forces. Additional in vitro and in vivo data show that 33PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus 34 retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase 35 is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be 36 broadly applicable to other type IVa pilus systems. 37 38Author Summary 39 Almost all bacterial species use thin surface appendages called pili to interact with their 40 environments. These structures are critical for the virulence of many pathogens and represent 41 one major way that bacteria share DNA with one another, which contributes to the spread of 42 antibiotic resistance. To carry out their function, pili dynamically extend and retract from the 43 bacterial surface. Here, we show that retraction of pili in some systems is determined by the 44 combined activity of two motor ATPase proteins. 45 46Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse 48 activities including attachment, virulence, biofilm formation, horizontal gene transfer, and 49 twitching motility [1][2][3][4][5]. These structures can dynamically extend and retract from the cell 50 surface, which is often critical for their function. A detailed mechanistic understanding of pilus 51 dynamic activity, however, remains lacking. 52Type IV pili are composed almost exclusively of a single protein called the major pilin, which 53 forms a helical fiber that extends from the cell surface [6]. Extension and retraction of the pilus is 54 hypothesized to occur through the interaction of cytoplasmic hexameric ATPases with the inner 55 membrane platform of the pilus machine, whereby ATP ...

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 53%

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Chlebek

Hughes

Ratkiewicz

et al. 2019

Preprint

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“…Optical tweezers, atomic force microscopy, micropillar assay and traction force microscopy are techniques to measure pilus retraction forces and also yield information about retraction dynamics, but in an indirect way and only for pilus retraction 4,16–21 . A recent study used interferometric imaging to directly image pili in living cells, but this technique generates a strong halo around the cell that overshadows any pili that are shorter than ~3 microns 22 . Despite the limitations of these approaches, they have led to several competing models for how the switch between TFP extension and retraction is controlled.…”

Section: Introductionmentioning

confidence: 99%

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Koch

Fei

Wingreen

et al. 2020

Preprint

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10The functions of type IV pili (TFP) are mediated by cycles of extension and retraction. The 11 coordination of these cycles remains mysterious due to poor quantification of TFP dynamics. Here we 12 fluorescently label the TFP in the opportunistic pathogen Pseudomonas aeruginosa and track the full 13 extension and retraction cycles of individual TFP to quantify their dynamics. We test several models for 14 the switch between extension and retraction using quantitative experiments, biophysical modeling and 15 genetics. We invalidate the prominent hypothesis that this switch is triggered by surface contact. Instead, 16 we show that the entire repetitive cycle of extension and retraction of individual TFP is governed by the 17 stochastic binding of antagonistic extension and retraction motors and explain how this mechanism 18 quantitatively defines physiologically-important features like TFP length and their production rate. 19Interestingly, our results suggest that the major throttle of TFP production is the unbinding of the 20 retraction motor. 21 22 microns 22 . Despite the limitations of these approaches, they have led to several competing models for 42 how the switch between TFP extension and retraction is controlled. A cryo-EM study did not observe 43 motors at the base of unpiliated structures, suggesting that the motors do not remain bound after TFP 44 retraction 12 . Meanwhile, an interferometry study focusing on the longest subpopulation of TFP suggested 45 that TFP retraction is triggered by surface association 22 . However, the inability to directly visualize the 46 dynamics of the entire TFP population previously limited the ability to directly test these models. 47Here we addressed the above limitations by directly fluorescently labeling the TFP of P. 48 aeruginosa. Fluorescent labeling of TFP was first achieved with non-specific labeling of extracellular 49 proteins 23 . However, similar to the interferometry approach, this surface labeling approach led to a strong 50 halo from staining of the cell body that prevented analysis of short pili. More recently, TFP from 51Caulobacter crescentus and Vibrio cholerae were directly labeled by introducing a reactive cysteine 52 residue into the pilin sequence 24-26 . Here we apply this approach to P. aeruginosa and use it to perform 53 the first direct quantitative analysis of full TFP extension and retraction cycles of individual pili. We go 54 on to develop and test quantitative models for the behaviors we observe. We show that TFP production 55 rate, length, and dynamics can be fully explained by the mutually exclusive stochastic binding of the 56 extension and retraction motors, and that this stochasticity persists in the presence or absence of surface 57 association. 58 59 Results 60Quantifying TFP dynamics reveals that P. aeruginosa makes mostly short pili that are highly dynamic 61We fluorescently labeled the major protein of the P. aeruginosa pilus fiber (PilA) by introducing 62 a cysteine point mutation, A86C, that we then labeled with the thiol-r...

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“…The EMBO Journal 38: e102145 | 2019 ª 2019 The Authors persistence length measured for P. aeruginosa TFP is longer than the sheared pili used here that are typically 2 lm in length (Skerker & Berg, 2001;Tala et al, 2019). It takes a liquid flow with a shear stress of a few dyn cm À2 to bend the pilus to adhere to the cellular surface, thus allowing a second form of adhesion.…”

Section: Of 16mentioning

confidence: 87%

“…Finally, direct microscopic visualization of the adhesion of purified pili to human cells shows that they bind cells via their distal tip. Interestingly, a tip-mediated mechanism of binding has previously been reported in P. aeruginosa by imaging fluorescently labeled pili of live bacteria adhering to quartz (Skerker & Berg, 2001;Tala et al, 2019).…”

Section: Of 16mentioning

confidence: 91%

Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip‐mediated adhesion

et al. 2019

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Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

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Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements

Cited by 132 publications

References 34 publications

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip‐mediated adhesion

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