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

2021 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Triana N. Dalia ◽  
Nicolas Biais ◽  
Ankur B. Dalia
Keyword(s):  
Cell Surface ◽  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Ectopic Expression ◽  
Type Iv Pili ◽  
Therapeutic Interventions ◽  
Dynamic Activity ◽  
Type Iv ◽  
Additional Support

ABSTRACTBacteria 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.

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

2021 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Triana N. Dalia ◽  
Nicolas Biais ◽  
Ankur B. Dalia
Keyword(s):  
Cell Surface ◽  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Ectopic Expression ◽  
Type Iv Pili ◽  
Therapeutic Interventions ◽  
Dynamic Activity ◽  
Type Iv ◽  
Additional Support

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. IMPORTANCE 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.

scholarly journals Type IV competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake

2021 ◽  
Author(s):  
Trinh Lam ◽  
Courtney K. Ellison ◽  
Ankur B. Dalia ◽  
David T. Eddington ◽  
Donald A. Morrison
Keyword(s):  
Cell Surface ◽  
Active Role ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Gram Positive ◽  
Dynamic Activity ◽  
Positive Type ◽  
Type Iv ◽  
Labeling Approach ◽  
Dynamic Structures

SUMMARYThe competence pili of transformable Gram-positive species form a subset of the diverse and widespread class of extracellular filamentous organelles known as type IV pili (T4P). In Gram-negative bacteria, T4P act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether T4P in Gram-positive species exhibit this same dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) passively form transient cavities in the cell wall to facilitate DNA passage, (2) act as static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via their dynamic activity. Here, using a recently described pilus labeling approach, we demonstrate that pneumococcal competence pili are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling ComGC with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results indicate that Gram-positive type IV competence pili are dynamic and retractile structures that play an active role in DNA uptake.Short summaryCompetent pneumococci kill non-competent cells on contact. Retractable DNA-binding fibers in the class of type IV pili may provide a key tool for retrieving DNA segments from cell wreckage for internalization and recombination.

scholarly journals Involvement of Type IV Pili in Pathogenicity of Plant Pathogenic Bacteria

Genes ◽  
2011 ◽  
Vol 2 (4) ◽  
pp. 706-735 ◽  
Author(s):  
Saul Burdman ◽  
Ofir Bahar ◽  
Jennifer K. Parker ◽  
Leonardo De La Fuente
Keyword(s):  
Pathogenic Bacteria ◽  
Type Iv Pili ◽  
Plant Pathogenic Bacteria ◽  
Type Iv

scholarly journals FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili

2006 ◽  
Vol 188 (13) ◽  
pp. 4851-4860 ◽  
Author(s):  
Sophie de Bentzmann ◽  
Marianne Aurouze ◽  
Geneviève Ball ◽  
Alain Filloux
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Type Iv Pili ◽  
Reading Frame ◽  
Type Iv ◽  
Bacterial Aggregation ◽  
Epithelial Cell Surface ◽  
Prepilin Peptidase ◽  
Pilus Assembly

ABSTRACT Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.

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

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Triana N. Dalia ◽  
Jennifer L. Chlebek ◽  
Ankur B. Dalia
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Industrial Applications ◽  
Model System ◽  
Promoter Element ◽  
Reporter System ◽  
Dynamic Activity ◽  
Type Iv ◽  
Ectopic Gene Expression ◽  
Integrated Platform

Abstract 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 Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-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.

scholarly journals Role ofCaulobacterCell Surface Structures in Colonization of the Air-Liquid Interface

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Aretha Fiebig
Keyword(s):  
Cell Surface ◽  
Caulobacter Crescentus ◽  
Three Dimensional ◽  
Liquid Interface ◽  
Type Iv Pili ◽  
Surface Structures ◽  
Aquatic Environments ◽  
Content Type ◽  
Type Iv ◽  
Air Liquid Interface

ABSTRACTIn aquatic environments,Caulobacterspp. can be found at the boundary between liquid and air known as the neuston. I report an approach to study temporal features ofCaulobacter crescentuscolonization and pellicle biofilm development at the air-liquid interface and have defined the role of cell surface structures in this process. At this interface,C. crescentusinitially forms a monolayer of cells bearing a surface adhesin known as the holdfast. When excised from the liquid surface, this monolayer strongly adheres to glass. The monolayer subsequently develops into a three-dimensional structure that is highly enriched in clusters of stalked cells known as rosettes. As this pellicle film matures, it becomes more cohesive and less adherent to a glass surface. A mutant strain lacking a flagellum does not efficiently reach the surface, and strains lacking type IV pili exhibit defects in organization of the three-dimensional pellicle. Strains unable to synthesize the holdfast fail to accumulate at the boundary between air and liquid and do not form a pellicle. Phase-contrast images support a model whereby the holdfast functions to trapC. crescentuscells at the air-liquid boundary. Unlike the holdfast, neither the flagellum nor type IV pili are required forC. crescentusto partition to the air-liquid interface. While it is well established that the holdfast enables adherence to solid surfaces, this study provides evidence that the holdfast has physicochemical properties that allow partitioning of nonmotile mother cells to the air-liquid interface and facilitate colonization of this microenvironment.IMPORTANCEIn aquatic environments, the boundary at the air interface is often highly enriched with nutrients and oxygen. Colonization of this niche likely confers a significant fitness advantage in many cases. This study provides evidence that the cell surface adhesin known as a holdfast enablesCaulobacter crescentusto partition to and colonize the air-liquid interface. Additional surface structures, including the flagellum and type IV pili, are important determinants of colonization and biofilm formation at this boundary. Considering that holdfast-like adhesins are broadly conserved inCaulobacterspp. and other members of the diverse classAlphaproteobacteria, these surface structures may function broadly to facilitate colonization of air-liquid boundaries in a range of ecological contexts, including freshwater, marine, and soil ecosystems.

scholarly journals Structure and function of minor pilins of type IV pili

2019 ◽  
Vol 209 (3) ◽  
pp. 301-308 ◽  
Author(s):  
Theis Jacobsen ◽  
Benjamin Bardiaux ◽  
Olivera Francetic ◽  
Nadia Izadi-Pruneyre ◽  
Michael Nilges
Keyword(s):  
Pathogenic Bacteria ◽  
Type Iv Pili ◽  
Host Cells ◽  
Type Ii Secretion ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Flexible Fibers ◽  
Multiple Copies ◽  
Species Specific ◽  
And Function

AbstractType IV pili are versatile and highly flexible fibers formed on the surface of many Gram-negative and Gram-positive bacteria. Virulence and infection rate of several pathogenic bacteria, such as Neisseria meningitidis and Pseudomonas aeruginosa, are strongly dependent on the presence of pili as they facilitate the adhesion of the bacteria to the host cell. Disruption of the interactions between the pili and the host cells by targeting proteins involved in this interaction could, therefore, be a treatment strategy. A type IV pilus is primarily composed of multiple copies of protein subunits called major pilins. Additional proteins, called minor pilins, are present in lower abundance, but are essential for the assembly of the pilus or for its specific functions. One class of minor pilins is required to initiate the formation of pili, and may form a complex similar to that identified in the related type II secretion system. Other, species-specific minor pilins in the type IV pilus system have been shown to promote additional functions such as DNA binding, aggregation and adherence. Here, we will review the structure and the function of the minor pilins from type IV pili.

scholarly journals Direct Regulation by the Vibrio cholerae Regulator ToxT To Modulate Colonization and Anticolonization Pilus Expression

2009 ◽  
Vol 77 (4) ◽  
pp. 1383-1388 ◽  
Author(s):  
Ansel Hsiao ◽  
Xiao Xu ◽  
Biao Kan ◽  
Rahul V. Kulkarni ◽  
Jun Zhu
Keyword(s):  
Transcription Factor ◽  
Vibrio Cholerae ◽  
Transcriptional Repression ◽  
Type Iv Pili ◽  
Present Evidence ◽  
Type Iv ◽  
Transcriptional Regulatory ◽  
Environmental Reservoir ◽  
Target Promoters ◽  
Direct Regulation

ABSTRACT The pathogen Vibrio cholerae uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host and establish itself at its preferred colonization site at the host intestinal mucosa. The key regulator in this process is the AraC/XylS family transcription factor, ToxT, which plays critical roles in pathogenesis, including the regulation of two type IV pili, the anticolonization factor mannose-sensitive hemagglutinin and the toxin-coregulated pilus. Previously, it was thought ToxT required dimerization in order to effect transcriptional regulation at its cognate promoters. Here, we present evidence that ToxT directly represses transcription of the msh operon by binding to three promoters within this operon and that dimerization may not be required for transcriptional repression of target promoters by ToxT, suggesting that this regulator uses different mechanisms to modulate the transcriptional repertoire of V. cholerae.

scholarly journals Expression of Type IV Pili by Moraxella catarrhalis Is Essential for Natural Competence and Is Affected by Iron Limitation

2004 ◽  
Vol 72 (11) ◽  
pp. 6262-6270 ◽  
Author(s):  
Nicole R. Luke ◽  
Amy J. Howlett ◽  
Jianqiang Shao ◽  
Anthony A. Campagnari
Keyword(s):  
Moraxella Catarrhalis ◽  
Pathogenic Bacteria ◽  
Type Iv Pili ◽  
Protein Subunit ◽  
Electron Microscopic ◽  
Type Iv ◽  
Wide Range ◽  
Genes Encoding ◽  
Microscopic Studies

ABSTRACT Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.

scholarly journals Discovery of a new Neisseria gonorrhoeae Type IV pilus assembly factor, TfpC

2020 ◽  
Author(s):  
Linda I. Hu ◽  
Shaohui Yin ◽  
Egon A. Ozer ◽  
Lee Sewell ◽  
Saima Rehman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Neisseria Gonorrhoeae ◽  
Sexually Transmitted Infection ◽  
Bacterial Cell ◽  
Bacterial Species ◽  
Type Iv Pili ◽  
Type Iv Pilus ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Type Iv

AbstractNeisseria gonorrhoeae rely on Type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection, gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the Type IV pilus in its extended, non-retracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show there are orthologues in numerous bacteria species but not all Type IV pilin expressing bacteria contain orthologous genes. Co-evolution and NMR analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region and a highly charge C-terminal coiled-coil domain.ImportanceMost bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae Type IV pilus is a major virulence and colonization factor for the sexually transmitted infection, gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain the Type IV pili on the bacterial cell surface. There are similar proteins found in the other members of the Neisseria genus and many other bacterial species important for human health.

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