Publications by authors named "Nicolas Biais"

38 Publications

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

Evolutionary algorithms converge towards evolved biological photonic structures.

Sci Rep 2020 07 21;10(1):12024. Epub 2020 Jul 21.

Université Clermont Auvergne, CNRS, Sigma Clermont, Institut Pascal, 63000, Clermont-Ferrand, France.

Nature features a plethora of extraordinary photonic architectures that have been optimized through natural evolution in order to more efficiently reflect, absorb or scatter light. While numerical optimization is increasingly and successfully used in photonics, it has yet to replicate any of these complex naturally occurring structures. Using evolutionary algorithms inspired by natural evolution and performing particular optimizations (maximize reflection for a given wavelength, for a broad range of wavelength or maximize the scattering of light), we have retrieved the most stereotypical natural photonic structures. Whether those structures are Bragg mirrors, chirped dielectric mirrors or the gratings on top of Morpho butterfly wings, our results indicate how such regular structures might have spontaneously emerged in nature and to which precise optical or fabrication constraints they respond. Comparing algorithms show that recombination between individuals, inspired by sexual reproduction, confers a clear advantage that can be linked to the fact that photonic structures are fundamentally modular: each part of the structure has a role which can be understood almost independently from the rest. Such an in silico evolution also suggests original and elegant solutions to practical problems, as illustrated by the design of counter-intuitive anti-reflective coatings for solar cells.
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http://dx.doi.org/10.1038/s41598-020-68719-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374560PMC
July 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

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

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium .

J Biol Chem 2019 04 5;294(17):6796-6808. Epub 2019 Mar 5.

From the Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom,

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, In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in 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, a methylated N terminus; (iii) are found in the same heteropolymeric 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 Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that 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.
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http://dx.doi.org/10.1074/jbc.RA118.006917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497953PMC
April 2019

Pili mediated intercellular forces shape heterogeneous bacterial microcolonies prior to multicellular differentiation.

Sci Rep 2018 11 8;8(1):16567. Epub 2018 Nov 8.

Brooklyn College of CUNY, Department of Biology, Brooklyn, USA.

Microcolonies are aggregates of a few dozen to a few thousand cells exhibited by many bacteria. The formation of microcolonies is a crucial step towards the formation of more mature bacterial communities known as biofilms, but also marks a significant change in bacterial physiology. Within a microcolony, bacteria forgo a single cell lifestyle for a communal lifestyle hallmarked by high cell density and physical interactions between cells potentially altering their behaviour. It is thus crucial to understand how initially identical single cells start to behave differently while assembling in these tight communities. Here we show that cells in the microcolonies formed by the human pathogen Neisseria gonorrhoeae (Ng) present differential motility behaviors within an hour upon colony formation. Observation of merging microcolonies and tracking of single cells within microcolonies reveal a heterogeneous motility behavior: cells close to the surface of the microcolony exhibit a much higher motility compared to cells towards the center. Numerical simulations of a biophysical model for the microcolonies at the single cell level suggest that the emergence of differential behavior within a multicellular microcolony of otherwise identical cells is of mechanical origin. It could suggest a route toward further bacterial differentiation and ultimately mature biofilms.
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http://dx.doi.org/10.1038/s41598-018-34754-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224386PMC
November 2018

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae.

Nat Microbiol 2018 07 11;3(7):773-780. Epub 2018 Jun 11.

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

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.
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http://dx.doi.org/10.1038/s41564-018-0174-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582970PMC
July 2018

Obstruction of pilus retraction stimulates bacterial surface sensing.

Science 2017 10;358(6362):535-538

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

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.
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http://dx.doi.org/10.1126/science.aan5706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805138PMC
October 2017

Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates.

New J Phys 2017 Jan 10;19(1). Epub 2017 Jan 10.

Max Planck Institute for the Physics of Complex Systems, D-01187 Dresden, Germany.

is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.
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http://dx.doi.org/10.1088/1367-2630/aa5483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132470PMC
January 2017

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

PLoS Pathog 2016 Dec 19;12(12):e1006109. Epub 2016 Dec 19.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.
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http://dx.doi.org/10.1371/journal.ppat.1006109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207764PMC
December 2016

Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis.

Nat Microbiol 2016 Jan 18;1:15004. Epub 2016 Jan 18.

Department of Biological Sciences, University of Notre Dame, 100 Galvin Life Sciences Center, Notre Dame, Indiana 46556, USA.

Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections(1). For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl(-) ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS.
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http://dx.doi.org/10.1038/nmicrobiol.2015.4DOI Listing
January 2016

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse.

Int J Syst Evol Microbiol 2016 Sep 13;66(9):3585-3593. Epub 2016 Jun 13.

Department of Immunobiology and BIO5 Institute, University of Arizona, Tucson, AZ 85719, USA.

Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.
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http://dx.doi.org/10.1099/ijsem.0.001237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880574PMC
September 2016

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

Cell 2016 Mar 25;165(1):100-110. Epub 2016 Feb 25.

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.
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http://dx.doi.org/10.1016/j.cell.2016.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808403PMC
March 2016

An In Vitro Model System to Test Mechano-microbiological Interactions Between Bacteria and Host Cells.

Methods Mol Biol 2016 ;1365:195-212

Department of Biology, Brooklyn College of the City University of New York, 307 Ingersoll Hall Extension, 2900 Bedford Avenue, Brooklyn, NY, 11210, USA.

The aim of this chapter is to present an innovative technique to visualize changes of the f-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust f-actin accumulation at the sites where pulling forces were applied. Using the magnetic tweezers we were able to mimic the local response of the f-actin cytoskeleton to bacteria-generated forces. In this chapter we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.
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http://dx.doi.org/10.1007/978-1-4939-3124-8_10DOI Listing
August 2016

Formation and dissolution of bacterial colonies.

Phys Rev E Stat Nonlin Soft Matter Phys 2015 Sep 3;92(3):032704. Epub 2015 Sep 3.

Max Planck Institute for the Physics of Complex Systems, Nöthnitzer Str. 38, Dresden 01187, Germany.

Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.
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http://dx.doi.org/10.1103/PhysRevE.92.032704DOI Listing
September 2015

Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis.

Mol Microbiol 2016 Jan 27;99(2):380-92. Epub 2015 Oct 27.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

Type IV pili (Tfp), which have been studied extensively in a few Gram-negative species, are the paradigm of a group of widespread and functionally versatile nano-machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface-associated motility. Tfp power 'train-like' directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano-machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram-negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano-machines.
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http://dx.doi.org/10.1111/mmi.13237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832360PMC
January 2016

Pili-Induced Clustering of N. gonorrhoeae Bacteria.

PLoS One 2015 10;10(9):e0137661. Epub 2015 Sep 10.

Harvard University, School of Engineering and Applied Sciences, Cambridge, MA, United States of America; Max-Planck-Institute for the Physics of Complex Systems, Dresden, Germany.

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137661PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565587PMC
May 2016

Common Cell Shape Evolution of Two Nasopharyngeal Pathogens.

PLoS Genet 2015 Jul 10;11(7):e1005338. Epub 2015 Jul 10.

Institut Pasteur, Groupe Biologie et Génétique de la Paroi Bactérienne, Département de Microbiologie, Paris, France; INSERM, Groupe Avenir, Paris, France.

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.
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http://dx.doi.org/10.1371/journal.pgen.1005338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498754PMC
July 2015

Mechanotransduction: use the force(s).

BMC Biol 2015 Jul 4;13:47. Epub 2015 Jul 4.

Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK.

Mechanotransduction - how cells sense physical forces and translate them into biochemical and biological responses - is a vibrant and rapidly-progressing field, and is important for a broad range of biological phenomena. This forum explores the role of mechanotransduction in a variety of cellular activities and highlights intriguing questions that deserve further attention.
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http://dx.doi.org/10.1186/s12915-015-0150-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491211PMC
July 2015

Analyzing bacterial movements on surfaces.

Methods Cell Biol 2015 8;125:453-69. Epub 2015 Jan 8.

Brooklyn College CUNY, Biology Department, Brooklyn, NY, USA; Graduate Center of CUNY, New York, NY, USA.

Bacteria have long been ideal model systems for studying many biological phenomena. But when it comes to motility, we are quite often just figuring out the mechanisms underlying their ability to move in liquid or on surfaces. In the last few decades, research has emphasized the importance for bacteria to be able to adhere to and move on surfaces in order to form complex bacterial communities called biofilms. To better understand the multiple chemical and biophysical mechanisms responsible for the initial interactions of bacteria on surfaces that develop into biofilms, we present here low-cost and easy-to-implement protocols to quantitatively analyze the movement of single bacteria on surfaces by microscopy. These protocols are presented in the case of the human pathogen Neisseria gonorrhoeae that moves on surfaces solely powered by Type IV pili, motility referred to as twitching motility. These methods, however, are applicable for any motile bacteria interacting with surfaces. The precise quantification of motility coupled with genetic tools will enable us to precisely dissect the mechanisms and dynamics of bacterial surface motility which are still poorly understood.
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http://dx.doi.org/10.1016/bs.mcb.2014.10.014DOI Listing
September 2015

Uncovering the mechanism of trapping and cell orientation during Neisseria gonorrhoeae twitching motility.

Biophys J 2014 Oct;107(7):1523-31

School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts; Department of Physics, Harvard University, Cambridge, Massachusetts. Electronic address:

Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces.
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http://dx.doi.org/10.1016/j.bpj.2014.07.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190650PMC
October 2014

Steered molecular dynamics simulations of a type IV pilus probe initial stages of a force-induced conformational transition.

PLoS Comput Biol 2013 Apr 11;9(4):e1003032. Epub 2013 Apr 11.

Department of Physics, University of Arizona, Tucson, Arizona, USA.

Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin α1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P's structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin α1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching.
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http://dx.doi.org/10.1371/journal.pcbi.1003032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623709PMC
April 2013

Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation.

Proc Natl Acad Sci U S A 2013 Apr 20;110(15):E1361-70. Epub 2013 Mar 20.

Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain.

Focal adhesions are mechanosensitive elements that enable mechanical communication between cells and the extracellular matrix. Here, we demonstrate a major mechanosensitive pathway in which α-actinin triggers adhesion maturation by linking integrins to actin in nascent adhesions. We show that depletion of the focal adhesion protein α-actinin enhances force generation in initial adhesions on fibronectin, but impairs mechanotransduction in a subsequent step, preventing adhesion maturation. Expression of an α-actinin fragment containing the integrin binding domain, however, dramatically reduces force generation in depleted cells. This behavior can be explained by a competition between talin (which mediates initial adhesion and force generation) and α-actinin for integrin binding. Indeed, we show in an in vitro assay that talin and α-actinin compete for binding to β3 integrins, but cooperate in binding to β1 integrins. Consistently, we find opposite effects of α-actinin depletion and expression of mutants on substrates that bind β3 integrins (fibronectin and vitronectin) versus substrates that only bind β1 integrins (collagen). We thus suggest that nascent adhesions composed of β3 integrins are initially linked to the actin cytoskeleton by talin, and then α-actinin competes with talin to bind β3 integrins. Force transmitted through α-actinin then triggers adhesion maturation. Once adhesions have matured, α-actinin recruitment correlates with force generation, suggesting that α-actinin is the main link transmitting force between integrins and the cytoskeleton in mature adhesions. Such a multistep process enables cells to adjust forces on matrices, unveiling a role of α-actinin that is different from its well-studied function as an actin cross-linker.
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http://dx.doi.org/10.1073/pnas.1220723110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625291PMC
April 2013

Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer.

Proc Natl Acad Sci U S A 2013 Feb 4;110(8):3059-64. Epub 2013 Feb 4.

BIO5 Institute and Department of Immunobiology, University of Arizona, Tucson, AZ 85721, USA.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.
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http://dx.doi.org/10.1073/pnas.1217420110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581930PMC
February 2013

Techniques to measure pilus retraction forces.

Methods Mol Biol 2012 ;799:197-216

Department of Biological Sciences, Columbia University, New York, NY, USA.

The importance of physical forces in biology is becoming more appreciated. Neisseria gonorrhoeaehas become a paradigm for the study of physical forces in the bacterial world. Cycles of elongations and retractions of Type IV pili enables N. gonorrhoeaebacteria to exert forces on its environment, forces that play major roles in the life cycle of this pathogen. In order to better understand the role of these forces, there is a need to fully characterize them. Here, we present two different techniques, optical tweezers and Polyacrylamide MicroPillars (PoMPs), for measuring pilus retraction forces. Initially designed for N. gonorrhoeae, these assays can be readily modified to study other pilus-bearing bacteria including Neisseria meningitidis.
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http://dx.doi.org/10.1007/978-1-61779-346-2_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5160128PMC
February 2012

N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae.

PLoS One 2011 22;6(6):e21373. Epub 2011 Jun 22.

Department of Immunobiology and the BIO5 Institute, University of Arizona, Tucson, Arizona, United States of America.

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021373PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120873PMC
November 2011

Magnetic micropillars as a tool to govern substrate deformations.

Lab Chip 2011 Aug 15;11(15):2630-6. Epub 2011 Jun 15.

Laboratoire Matière et Systèmes Complexes (MSC), Université Paris-Diderot & CNRS UMR 7057, Bâtiment Condorcet, Paris, France.

Magnetic actuated microdevices can be used to achieve several complex functions in microfluidics and microfabricated devices. For example, magnetic mixers and magnetic actuators have been proposed to help handling fluids at a small scale. Here, we present a strategy to create magnetically actuated micropillar arrays. We combined microfabrication techniques and the dispersion of magnetic aggregates embedded inside polymeric matrices to design micrometre scale magnetic features. By creating a magnetic field gradient in the vicinity of the substrate, well-defined forces were applied on these magnetic aggregates which in turn induced a deflection of the micropillars. By dispersing either spherical aggregates or magnetic nanowires into the gels, we can induce synchronized motions of a group of pillars or the movement of isolated pillars under a magnetic field gradient. When combined with microfabrication processes, this versatile tool leads to local as well as global substrate actuations within a range of dimensions that are relevant for microfluidics and biological applications.
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http://dx.doi.org/10.1039/c1lc20263dDOI Listing
August 2011

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions.

Mol Biol Cell 2011 Apr 16;22(8):1263-73. Epub 2011 Feb 16.

Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.
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http://dx.doi.org/10.1091/mbc.E10-08-0661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198308PMC
April 2011

Force-dependent polymorphism in type IV pili reveals hidden epitopes.

Proc Natl Acad Sci U S A 2010 Jun 3;107(25):11358-63. Epub 2010 Jun 3.

Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027, USA.

Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.
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http://dx.doi.org/10.1073/pnas.0911328107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895099PMC
June 2010
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