scholarly journals More than a feeling: microscopy approaches to understanding surface-sensing mechanisms

2020 ◽  
Author(s):  
Katherine J. Graham ◽  
Lori L. Burrows
Keyword(s):  
Live Cells ◽  
Surface Attachment ◽  
Bacterial Surface ◽  
Structural Modifications ◽  
Structure And Dynamics ◽  
Type Iv ◽  
Surface Sensing ◽  
Two Component Signal Transduction ◽  
Sense And Respond

The mechanisms by which bacteria sense and respond to surface attachment have long been a mystery. Our understanding of the structure and dynamics of bacterial appendages, notably type IV pili (T4P), provided new insights into the potential ways that bacteria sense surfaces. T4P are ubiquitous, retractable hair-like adhesins that until recently were difficult to image in the absence of fixation due to their nanoscale size. This review focuses on recent microscopy innovations used to visualize T4P in live cells to reveal the dynamics of their retraction and extension. We discuss recently proposed mechanisms by which T4P facilitate bacterial surface sensing, including the role of surface-exposed PilY1, two-component signal transduction pathways, force-induced structural modifications of the major pilin, and altered dynamics of the T4P motor complex.

scholarly journals Obstruction of pilus retraction stimulates bacterial surface sensing

2017 ◽  
Author(s):  
Courtney K. Ellison ◽  
Jingbo Kan ◽  
Rebecca S. Dillard ◽  
David T. Kysela ◽  
Cheri M. Hampton ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Caulobacter Crescentus ◽  
Physiological Changes ◽  
Type Iv Pilus ◽  
Dynamic Nature ◽  
Surface Attachment ◽  
Bacterial Surface ◽  
Type Iv ◽  
Surface Sensing ◽  
Surface Contact

AbstractSurface 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 thatCaulobacter crescentuspili 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.One Sentence SummaryBacteria use the tension imparted on retracting pilus fibers upon their binding to a surface for surface sensing.

scholarly journals Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection

2004 ◽  
Vol 72 (9) ◽  
pp. 5143-5149 ◽  
Author(s):  
Andreas B. den Hartigh ◽  
Yao-Hui Sun ◽  
David Sondervan ◽  
Niki Heuvelmans ◽  
Marjolein O. Reinders ◽  
...  
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Persistent Infection ◽  
Host Cells ◽  
Intracellular Replication ◽  
Bacterial Surface ◽  
Type Iv

ABSTRACT The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.

scholarly journals Tad pili play a dynamic role in Caulobacter crescentus surface colonization

2019 ◽  
Author(s):  
Matteo Sangermani ◽  
Isabelle Hug ◽  
Nora Sauter ◽  
Thomas Pfohl ◽  
Urs Jenal
Keyword(s):  
Caulobacter Crescentus ◽  
Optical Trap ◽  
Natural Environments ◽  
Surface Attachment ◽  
Surface Colonization ◽  
Bacterial Surface ◽  
Controlled Flow ◽  
Mechanical Sensing

ABSTRACTBacterial surface attachment is mediated by rotary flagella and filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions as a mimic of natural environments, we demonstrate that Tad pili undergo repeated cycles of extension and retraction. Within seconds after establishing surface contact, pili reorient cells into an upright position promoting walking-like movements against the medium flow. Pili-mediated positioning of the flagellated pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pili dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model, in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells towards permanent surface attachment.

scholarly journals Social Cooperativity of Bacteria during Reversible Surface Attachment in Young Biofilms: a Quantitative Comparison of Pseudomonas aeruginosa PA14 and PAO1

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Calvin K. Lee ◽  
Jérémy Vachier ◽  
Jaime de Anda ◽  
Kun Zhao ◽  
Amy E. Baker ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stochastic Model ◽  
Bacterial Biofilm ◽  
Population Increase ◽  
Pivotal Phase ◽  
Surface Attachment ◽  
Content Type ◽  
Cellular Behavior ◽  
Bacterial Surface ◽  
Surface Sensing

ABSTRACT What are bacteria doing during “reversible attachment,” the period of transient surface attachment when they initially engage a surface, besides attaching themselves to the surface? Can an attaching cell help any other cell attach? If so, does it help all cells or employ a more selective strategy to help either nearby cells (spatial neighbors) or its progeny (temporal neighbors)? Using community tracking methods at the single-cell resolution, we suggest answers to these questions based on how reversible attachment progresses during surface sensing for Pseudomonas aeruginosa strains PAO1 and PA14. Although PAO1 and PA14 exhibit similar trends of surface cell population increase, they show unanticipated differences when cells are considered at the lineage level and interpreted using the quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two surface colonization strategies. PAO1 lineages commit quickly to a surface compared to PA14 lineages, with early c-di-GMP-mediated exopolysaccharide (EPS) production that can facilitate the attachment of neighbors. PA14 lineages modulate their motility via cyclic AMP (cAMP) and retain memory of the surface so that their progeny are primed for improved subsequent surface attachment. Based on the findings of previous studies, we propose that the differences between PAO1 and PA14 are potentially rooted in downstream differences between Wsp-based and Pil-Chp-based surface-sensing systems, respectively. IMPORTANCE The initial pivotal phase of bacterial biofilm formation known as reversible attachment, where cells undergo a period of transient surface attachment, is at once universal and poorly understood. What is more, although we know that reversible attachment culminates ultimately in irreversible attachment, it is not clear how reversible attachment progresses phenotypically, as bacterial surface-sensing circuits fundamentally alter cellular behavior. We analyze diverse observed bacterial behavior one family at a time (defined as a full lineage of cells related to one another by division) using a unifying stochastic model and show that our findings lead to insights on the time evolution of reversible attachment and the social cooperative dimension of surface attachment in PAO1 and PA14 strains.

Two repetitive, biofilm-forming proteins from Staphylococci: from disorder to extension

2015 ◽  
Vol 43 (5) ◽  
pp. 861-866 ◽  
Author(s):  
Fiona Whelan ◽  
Jennifer R. Potts
Keyword(s):  
Medical Device ◽  
Staphylococcus Epidermidis ◽  
Molecular Mechanisms ◽  
Surface Proteins ◽  
Initial Surface ◽  
Surface Attachment ◽  
Bacterial Surface ◽  
Host Interactions ◽  
And Function

Staphylococcus aureus and Staphylococcus epidermidis are an important cause of medical device-related infections that are difficult to treat with antibiotics. Biofilms, in which bacteria are embedded in a bacterially-produced exopolymeric matrix, form on the surface of the implanted medical device. Our understanding of the molecular mechanisms underlying the initial surface attachment and subsequent intercellular interactions as the biofilm matures is improving. Biofilm accumulation can be mediated by a partially deacetylated form of poly-N-acetylglucosamine (PNAG) but, more recently, the role of bacterial surface proteins is being recognized. Here we describe the structure and function of two S. aureus cell surface proteins, FnBPA and SasG, implicated in host interactions and biofilm accumulation. These multifunctional proteins employ intrinsic disorder for distinct molecular outcomes. In the case of FnBPA, disorder generates adhesive arrays that bind fibronectin (Fn); in the case of SasG, disorder is, counterintuitively, used to maintain a strong extended fold.

scholarly journals The type IV pilin PilA couples surface attachment and cell cycle initiation in Caulobacter crescentus

2019 ◽  
Author(s):  
Luca Del Medico ◽  
Dario Cerletti ◽  
Matthias Christen ◽  
Beat Christen
Keyword(s):  
Cell Cycle ◽  
Biofilm Formation ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Peptide Mimetics ◽  
Cycle Progression ◽  
Surface Attachment ◽  
Type Iv ◽  
Surface Sensing ◽  
Type Iv Pilin

Understanding how bacteria colonize surfaces and regulate cell cycle progression in response to cellular adhesion is of fundamental importance. Here, we used transposon sequencing in conjunction with FRET microscopy to uncover the molecular mechanism how surface sensing drives cell cycle initiation in Caulobacter crescentus. We identified the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell cycle initiation via the second messenger c-di-GMP. Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17 amino-acid transmembrane helix of PilA to activate the PleC-PleD two component signaling system, increase cellular c-di-GMP levels and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for cell cycle initiating pilin peptide. Addition of the chemically synthesized CIP peptide initiates cell cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer new strategies to inhibit surface-sensing, prevent biofilm formation and control persistent infections.Significance StatementPili are hair-like appendages found on the surface of many bacteria to promote adhesion. Here, we provide systems-level findings on a molecular signal transduction pathway that interlinks surface sensing with cell cycle initiation. We propose that surface attachment induces depolymerization of pili filaments. The concomitant increase in pilin sub-units within the inner membrane function as a stimulus to activate the second messenger c-di-GMP and trigger cell cycle initiation. Further-more, we show that the provision of a 17 amino acid synthetic peptide corresponding to the membrane portion of the pilin sub-unit mimics surface sensing, activates cell cycle initiation and inhibits surface attachment. Thus, synthetic peptide mimetics of pilin may represent new chemotypes to control biofilm formation and treat bacterial infections.

scholarly journals Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes

2016 ◽  
Vol 198 (18) ◽  
pp. 2524-2535 ◽  
Author(s):  
Egidio Lacanna ◽  
Colette Bigosch ◽  
Volkhard Kaever ◽  
Alex Boehm ◽  
Anke Becker
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Primary Role ◽  
Bacterial Cells ◽  
Diguanylate Cyclase ◽  
Surface Attachment ◽  
Content Type ◽  
Surface Sensing

ABSTRACTDgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment ofEscherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription ofdgcZis regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of acpxRmutation and the positive effect ofnlpEoverexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface.IMPORTANCEBacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment.E. colipossesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes.

scholarly journals The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus

2020 ◽  
Vol 117 (17) ◽  
pp. 9546-9553 ◽  
Author(s):  
Luca Del Medico ◽  
Dario Cerletti ◽  
Philipp Schächle ◽  
Matthias Christen ◽  
Beat Christen
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Caulobacter Crescentus ◽  
Resonance Energy ◽  
Cellular Adhesion ◽  
Cycle Progression ◽  
Surface Attachment ◽  
Type Iv ◽  
Surface Sensing ◽  
Type Iv Pilin

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus. We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for “cell-cycle initiating pilin” peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

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

2019 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Hannah Q. Hughes ◽  
Aleksandra S. Ratkiewicz ◽  
Rasman Rayyan ◽  
Joseph Che-Yen Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Dependent Manner ◽  
Acinetobacter Baylyi ◽  
Bacterial Surface ◽  
Type Iv ◽  
Bona Fide ◽  
Almost All

AbstractBacterial 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 becausepilUmutants 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 ofVibrio choleraeas 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 ΔpilTstrains. Thus, these data suggest that PilU is abona fideretraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilUmutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additionalin vitroandin vivodata 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 inAcinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.Author 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.

scholarly journals A bifunctional ATPase drives tad pilus extension and retraction

2019 ◽  
Author(s):  
Courtney K. Ellison ◽  
Jingbo Kan ◽  
Jennifer L. Chlebek ◽  
Katherine R. Hummels ◽  
Gaёl Panis ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Atp Hydrolysis ◽  
Caulobacter Crescentus ◽  
Dynamic Activity ◽  
Surface Attachment ◽  
Type Iv ◽  
Surface Sensing ◽  
Domains Of Life ◽  
Macromolecular Protein

AbstractMolecular motors convert chemical energy directly into mechanical work1and 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 tightadherence (tad) pili are critical for surface sensing, surface attachment, and biofilm formation8–10. How tad pili undergo dynamic cycles of extension and retraction8despite 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 ofCaulobacter crescentusCpaF 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 ofVibrio choleraethat 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.

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