The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase

Dw, Adams; Pereira, Jorge A.; Stoudmann, Candice; Stutzmann, Sandrine; Blokesch, Melanie

doi:10.1371/journal.pgen.1008393

Cited by 37 publications

(54 citation statements)

References 81 publications

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“…Our results indicate that PilU can only facilitate pilus retraction in the presence of PilT, regardless of whether that PilT is a functional ATPase. These results are also supported by a recent report from the Blokesch lab, which was published while our manuscript was in revision [41]. We initially tested the hypothesis that this was due to a requirement for PilT to bridge PilU with the platform protein PilC.…”

Section: Discussionsupporting

confidence: 81%

“…cholerae competence pili ( Fig 1A and S1 Fig ). Additionally, it was very recently shown that PilU from Pseudomonas aeruginosa can also promote retraction in a PilT-dependent manner [41]. Together, these results demonstrate that PilU ATPase activity facilitates pilus retraction in a PilT-dependent manner in at least three gammaproteobacteria ( V .…”

Section: Resultsmentioning

confidence: 66%

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

et al. 2019

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Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.

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Section: Discussionsupporting

confidence: 81%

Section: Resultsmentioning

confidence: 66%

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

et al. 2019

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“…6), suggesting that PilU can only function as a retraction ATPase for the MSHA system in the presence of PilT. Similar results have recently been reported for MSHA and ChiRP pili for O1 El Tor strain A1552, as well as for ChiRP in strain E7946, suggesting that this PilT-dependent PilU function is similarly conserved across O1 El Tor isolates 37,38 . c-di-GMP levels mediate MSHA production and retraction.…”

Section: Msha Retraction Appears Common Among O1 El Tor Isolatessupporting

confidence: 80%

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

et al. 2020

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Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.

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“…Our analysis confirms that PilU is indeed not needed for retraction but does affect retraction speed (Supplementary Figure 6). Recent studies also suggest that these motors may have more complicated interactions 38,39 , and in the future our model could help tease apart the specific contributions of different mutants to the extension and retraction cycle.…”

Section: Discussionmentioning

confidence: 87%

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...

show abstract

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase

Cited by 37 publications

References 81 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

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

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

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