Publications by authors named "Jennifer L Chlebek"

6 Publications

  • Page 1 of 1

Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction.

Appl Environ Microbiol 2021 06 25;87(14):e0047821. Epub 2021 Jun 25.

Department of Biology, Indiana University, Bloomington, Indiana, USA.

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 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 remain 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. Extracellular 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 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.
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http://dx.doi.org/10.1128/AEM.00478-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231728PMC
June 2021

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae.

Sci Rep 2020 09 21;10(1):15398. Epub 2020 Sep 21.

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements P and P to versions that have an additional theophylline-responsive riboswitch (P-riboswitch and P-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with P > P > P-riboswitch > P-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for P > P-riboswitch > P; while the newly developed P-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible P-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.
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http://dx.doi.org/10.1038/s41598-020-72387-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505983PMC
September 2020

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

Nat Commun 2020 03 25;11(1):1549. Epub 2020 Mar 25.

Department of Microbiology and Environmental Toxicology, University of California - Santa Cruz, 1156 High St., BioMed 245, Santa Cruz, CA, 95064, USA.

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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http://dx.doi.org/10.1038/s41467-020-15331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096442PMC
March 2020

A bifunctional ATPase drives tad pilus extension and retraction.

Sci Adv 2019 12 18;5(12):eaay2591. Epub 2019 Dec 18.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA.

A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for surface sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract 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 adenosine 5'-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction.
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http://dx.doi.org/10.1126/sciadv.aay2591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920026PMC
December 2019

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae.

PLoS Genet 2019 10 28;15(10):e1008362. Epub 2019 Oct 28.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.
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http://dx.doi.org/10.1371/journal.pgen.1008362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855506PMC
October 2019

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

PLoS Genet 2019 10 18;15(10):e1008448. Epub 2019 Oct 18.

Department of Biology, Indiana University, Bloomington, Indiana, United States of America.

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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http://dx.doi.org/10.1371/journal.pgen.1008448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821130PMC
October 2019
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