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

Jennifer L Chlebek,Aleksandra S Ratkiewicz,Ankur B Dalia,Triana N Dalia,Hannah Q Hughes,Nicolas Biais,Rasman Rayyan,Joseph Che-Yen Wang,Brittany E Herrin,Danielle A Garsin

doi:10.1371/journal.pgen.1008448

Jennifer L Chlebek, Aleksandra S Ratkiewicz + Show 8 more

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https://doi.org/10.1371/journal.pgen.1008448

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Abstract

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.

Highlights

Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse activities including attachment, virulence, biofilm formation, horizontal gene transfer, and twitching motility [1,2,3,4,5]
These structures are critical for the virulence of many pathogens and represent one major way that bacteria share DNA with one another, which contributes to the spread of antibiotic resistance
We have recently demonstrated that the type IV competence pili of V. cholerae can be fluorescently labeled by using a technique [14,15,16] in which an amino acid of the major pilin subunit PilA is replaced with a cysteine (PilAS56C based on the mature pilin sequence, which was previously misannotated PilAS67C [17] and PilAS81C [18]), which allows for subsequent labeling with a fluorescently-labeled thiol-reactive maleimide dye (AF488-mal)

Summary

Introduction

Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse activities including attachment, virulence, biofilm formation, horizontal gene transfer, and twitching motility [1,2,3,4,5]. These structures can dynamically extend and retract from the cell surface, which is often critical for their function. Many type IVa pilus systems contain a retraction ATPase, called PilT, which depolymerizes the pilus fiber and recycles pilin subunits into the inner membrane. A pilU mutant of Pseudomonas aeruginosa, exhibits reduced twitching motility [13], but in other type IV pilus systems, including the V. cholerae competence pilus, loss of pilU does not reveal any overt phenotypes

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