Biology subjects

Hughes, H. Q.

Publications and source records attributed to Hughes, H. Q..

3 recordsLinked to original sources

bioRxiv2022

The canonical PilT retraction ATPase promotes both extension and retraction of the MSHA type IVa pilus in Vibrio cholerae

Diverse bacterial species use type IVa pili (T4aP) to interact with their environments. The dynamic extension and retraction of T4aP is critical for their function, but the mechanisms that regulate this dynamic activity remain poorly understood. T4aP are typically extended via the activity of a dedicated extension motor ATPase and retracted via the action of an antagonistic retraction motor ATPase called PilT. These motors are generally functionally independent, and loss of PilT commonly results in T4aP hyperpiliation due to undeterred pilus extension. However, for the mannose-sensitive hemagglutinin (MSHA) T4aP of Vibrio cholerae, the loss of PilT results in a loss of surface piliation, which is unexpected based on our current understanding of T4aP dynamics. Here, we employ a combination of genetic and cell biological approaches to dissect the underlying mechanism. Our results demonstrate that PilT is necessary for MSHA pilus extension in addition to its well-established role in promoting MSHA pilus retraction. Through a suppressor screen, we also provide genetic evidence that the MshA major pilin impacts pilus extension. Together, these findings contribute to our understanding of the factors that regulate pilus extension and describe a previously uncharacterized function for the PilT motor ATPase. AUTHOR SUMMARYMany bacteria use filamentous appendages called type IVa pili to interact with their environment. These fibers dynamically extend and retract through the activity of ATPase motor proteins. In most pilus systems, deletion of the retraction motor results in uninterrupted pilus extension, leading to hyperpiliation. However, in the MSHA pilus system of V. cholerae, deletion of the retraction motor, pilT, results in a decrease in the number of surface pili. Here, we show that PilT is unexpectedly required for MSHA pilus extension in addition to its defined role in promoting pilus retraction. These results extend our understanding of the complex mechanisms underlying the dynamic activity of these broadly conserved filamentous appendages.

microbiology↗

bioRxiv2021

Oxygen depletion and nitric oxide stimulate type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. Dynamic extension and retraction of pili is often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To study this question, we use the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we find that V. cholerae cells retract MSHA pili and detach from a surface in microaerobic conditions. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP (c-di-GMP) under microaerobic conditions to induce MSHA pilus retraction. CdpA contains a putative NO-sensing NosP domain, and we demonstrate that nitric oxide (NO) is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that microaerobic conditions result in endogenous production of NO (or an NO-like molecule) in V. cholerae. Together, these results describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. More broadly, these results show how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

microbiology↗

bioRxiv2019

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

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 {Delta}pilT 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 {Delta}pilU 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.\n\nAuthor SummaryAlmost all bacterial species use thin surface appendages called pili to interact with their environments. 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. To carry out their function, pili dynamically extend and retract from the bacterial surface. Here, we show that retraction of pili in some systems is determined by the combined activity of two motor ATPase proteins.

microbiology↗