Biology subjects

Biais, N.

Publications and source records attributed to Biais, N..

7 recordsLinked to original sources

bioRxiv2026

A short C-terminal plug motif in a conserved type 4 pilus component controls pilus-tip localisation and filament biogenesis

Type 4a pili (T4aP), the most widespread and functionally versatile subtype of the type 4 filaments (T4F) superfamily, are functionalised at their tip by the adhesin PilC/PilY1. How this unusually large non-pilin protein is stably displayed at the pilus tip, and why is it required for pilus biogenesis, remains unknown. Here, using a multidisciplinary approach in Neisseria meningitidis, we show that the last 12 residues of PilC, representing ~1% of the protein, are both necessary and sufficient for pilus-tip localisation and filament biogenesis. X-ray crystallography reveals that this short "plug" motif binds the PilK subunit (within a complex of four minor pilins that caps the pilus) through {beta}-strand augmentation, a mode of interaction not previously characterised in T4F. Binding assays with synthetic peptides establish the specificity and affinity of this interaction, while the addition of a plug peptide extracellularly to cultures of a{Delta} pilC mutant restores pilus biogenesis. We show, using different methods, that the plug motif markedly stabilises PilK, providing an explanation for the requirement of PilC in pilus biogenesis. Consequently, expression of a PilK protein carrying a fused plug in N. meningitidis bypasses the requirement for PilC in pilus biogenesis. Together, these findings define the molecular basis of PilC/PilY1 function and localisation, reconcile all previous observations, and establish a broadly applicable model for T4aP biogenesis.

microbiology↗

bioRxiv2024

Oxydifficidin, a potent Neisseria gonorrhoeae antibiotic due to DedA assisted uptake and ribosomal protein RplL sensitivity

Gonorrhea, which is caused by Neisseria gonorrhoeae, is the second most reported sexually transmitted infection worldwide. The increasing appearance of isolates that are resistant to approved therapeutics raises the concern that gonorrhea may become untreatable. Here, we serendipitously identified oxydifficidin as a potent N. gonorrhoeae antibiotic through the observation of a Bacillus amyloliquefaciens contaminant in a lawn of N. gonorrhoeae. Oxydifficidin is active against both wild- type and multidrug-resistant N. gonorrhoeae. Its potent activity results from a combination of DedA- assisted uptake into the cytoplasm and the presence of an oxydifficidin-sensitive ribosomal protein L7/L12 (RplL). Our data indicates that oxydifficidin binds to the ribosome at a site that is distinct from other antibiotics and that L7/L12 is uniquely associated with its mode of action. This study opens a potential new avenue for addressing antibiotic resistant gonorrhea and underscores the possibility of identifying overlooked natural products from cultured bacteria, particularly those with activity against previously understudied pathogens.

microbiology↗

bioRxiv2021

Fresh extension of Vibrio cholerae competence type IV pili predisposes them for motor-independent retraction

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. SIGNIFICANCEExtracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the external pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

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↗

bioRxiv2019

A bifunctional ATPase drives tad pilus extension and retraction

Molecular motors convert chemical energy directly into mechanical work1 and are found in all domains of life2. These motors are critical to intracellular transport3, motility4,5, macromolecular protein assembly3,6, and many essential processes7. A wide-spread class of related bacterial motors drive the dynamic activity of extracellular fibers, such as type IV pili (T4P), that are extended and retracted using so-called secretion motor ATPases. Among these, the tight adherence (tad) pili are critical for surface sensing, surface attachment, and biofilm formation8-10. How tad pili undergo dynamic cycles of extension and retraction8 despite lacking a dedicated retraction motor ATPase has remained a mystery. Here we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through ATP hydrolysis. Specifically, we show that mutations within the ATP hydrolysis active site of Caulobacter crescentus CpaF result in a correlated reduction in the rates of extension and retraction. Moreover, a decrease in the rate of ATP hydrolysis directly scales with a decrease in the force of retraction and reduced dynamics in these CpaF mutants. This mechanism of motor protein bifunctionality extends to another genus of tad-bearing bacteria. In contrast, the T4aP subclass of pili possess dedicated extension and retraction motor ATPase paralogs. We show that these processes are uncoupled using a slow ATP hydrolysis mutation in the extension ATPase of competence T4aP of Vibrio cholerae that decreases the rate of extension but has no effect on the rate of retraction. Thus, a single motor ATPase is able to drive the bidirectional processes of pilus fiber extension and retraction.

microbiology↗

bioRxiv2018

In depth biochemical and structural analysis of a Gram-positive type IV pilus

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N-terminus; (iii) are found in the same hetero-polymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin fold with a highly unusual flexible C-terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

microbiology↗

bioRxiv2017

Obstruction of pilus retraction stimulates bacterial surface sensing

Surface association provides numerous fitness advantages to bacteria. Thus, it is critical for bacteria to recognize surface contact and to consequently initiate physiological changes required for a surface-associated lifestyle (1). Ubiquitous microbial appendages called pili are involved in sensing surfaces and mediating downstream surface-associated behaviors (2-6). The mechanism by which pili mediate surface sensing remains unknown, largely due to the difficulty to visualize their dynamic nature and to directly modulate their activity without genetic modification. Here, we show that Caulobacter crescentus pili undergo dynamic cycles of extension and retraction that cease within seconds of surface contact, and this arrest of pilus activity coincides with surface-stimulated holdfast synthesis. By physically blocking pili, we show that imposing resistance to pilus retraction is sufficient to stimulate holdfast synthesis in the absence of surface contact. Thus, resistance to type IV pilus retraction upon surface attachment is used for surface sensing.\n\nOne Sentence SummaryBacteria use the tension imparted on retracting pilus fibers upon their binding to a surface for surface sensing.

microbiology↗