Mechanical force-induced morphology changes in a human fungal pathogen.

BACKGROUND: The initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion. RESULTS: In the present study, we have used PDMS micro-fabrication to probe the ability of filamentous C. albicans cells to penetrate and grow invasively in substrates of different stiffness. We show that there is a threshold for penetration that corresponds to a stiffness of ~ 200 kPa and that invasive growth within a stiff substrate is characterized by dramatic filament buckling, along with a stiffness-dependent decrease in extension rate. We observed a striking alteration in cell morphology, i.e., reduced cell compartment length and increased diameter during invasive growth, that is not due to depolarization of active Cdc42, but rather occurs at a substantial distance from the site of growth as a result of mechanical compression. CONCLUSIONS: Our data reveal that in response to this compression, active Cdc42 levels are increased at the apex, whereas active Rho1 becomes depolarized, similar to that observed in membrane protrusions. Our results show that cell growth and morphology are altered during invasive growth, suggesting stiffness dictates the host cells that C. albicans can penetrate.

Cavitation Mean Expectation Time in a Stretched Lennard-Jones Fluid under Confinement.

We investigate the nucleation of cavitation bubbles in a confined Lennard-Jones fluid subjected to negative pressures in a cubic enclosure. We perform molecular dynamics (MD) simulations with tunable interatomic potentials that enable us to control the wettability of solid walls by the liquid, that is, its contact angle. For a given temperature and pressure, as the solid is taken more hydrophobic, we put in evidence, an increase in nucleation probability. A Voronoi tessellation method is used to accurately detect the bubble appearance and its nucleation rate as a function of the contact angle. We adapt classical nucleation theory (CNT) proposed for the heterogeneous case on a flat surface to our situation where bubbles may appear on flat walls, edges, or corners of the confined box. We finally calculate a theoretical mean expectation time in these three cases. The ratio of these calculated values over the homogeneous case is computed and compared successfully against MD simulations. Beyond the infinite liquid case, this work explores the heterogeneous nucleation of cavitation bubbles, not only in the flat surface case but for more complex confining geometries.

Hybrid surfaces combining electropolymerization and lithography: fabrication and wetting properties.

In the current work, we developed a novel method to fabricate hybrid surfaces consisting of mixed hydrophilic/superhydrophobic properties. These surfaces specifically consist of a regular array of hydrophilic pillars (displaying a receding contact angle lower than 90°) surrounded by a superhydrophobic thinner layer made via the electropolymerization of a fluorinated monomer. Then, we determined the wetting properties of various forms of this complex surface, i.e., displaying different surface properties, by specifically determining their advancing (θa) and receding (θr) contact angles. Two main parameters were varied: the pillar density (from 21.2% to 6.5% based on using a spacing d between pillars varying from 25 to 45 micrometers) and the polymer charge density (from 0 to 100 mC cm-2). We observed that, for low charge density values, only the ground surface was covered by the hydrophobic polymers; while for higher charge density values, polymerization reached higher levels on the lateral surfaces of the nonconductive cylindrical pillars, eventually up to their top surfaces and covering them for the highest charge densities. This feature gave us an additional parameter that we could use to control the surface wettability. We also found that contact angles (advancing and receding) increased markedly with increasing polymer charge density above a critical value (which was higher for receding angles). And we measured advancing and receding contact angles to, respectively, increase and decrease with increasing pillar density. We interpreted qualitatively these behaviors, the main point being the importance of the impalement (null, partial or total).

Capillary bridge technique to study superhydrophobic surfaces.

We present here the use of the capillary bridge technique to study the wetting properties (advancing and receding contact angles) of transparent, textured and superhydrophobic surfaces over large wetted area. Apparent contact angles on such surfaces are classically measured using a goniometer in combination with video camera side visualization and a drop shape analysis. Recent experiments of Schellenberger et al. [F. Schellenberger, N. Encinas, D. Vollmer and H. J. Butt, Phys. Rev. Lett., 2016, 116(9), 096101] show that this method can significantly underestimate the apparent advancing contact angle. We use for the first time the capillary bridge setup for such textured surfaces, leading to a large (up to several cm2) wetted area, instead of having a reduced contact zone as in the drop case (mm2 or less). (1) We show here how to use the method and its characteristics to explore the wetting properties of superhydrophobic surfaces. We have developed a new analysis method in order to obtain the value of the contact angle for any position of the substrate. (2) We compare with the classical drop side view method, showing that advancing contact angles are systematically higher. (3) We compare to a few existing models, concluding a good agreement for receding values but not for advancing angles, for which models must be refined.

The detailed acoustic signature of a micro-confined cavitation bubble.

Numerous scenarios exist for a cavitation bubble growing in a liquid. We focus here on cavitation phenomena within water under static tension in a confined environment. Drawing inspiration from the natural materials in plants, we design a novel experimental setup where a micrometric volume of water is confined by a hydrogel-based material. We show that, submerging the sample in a hypertonic solution, the water within the cavity is placed under tension and the acoustic emission produced by the resulting bubble nucleation is precisely detected. This new experimental procedure is able to strongly reduce the acoustic reflections occurring at the hydrogel/air interface with more classical techniques. We also propose a mathematical model to characterise the pressure wave emitted in order to correctly take into account the dissipation effect induced by the visco-elastic behaviour of the confining hydrogel. Both bubble resonant frequency and damping are captured by the model and quantitatively match the values found in the experiments.

Automated high-content image-based characterization of microorganism behavioral diversity and distribution.

Microorganisms have evolved complex systems to respond to environmental signals. Gradients of particular molecules and elemental ions alter the behavior of microbes and their distribution within their environment. Microdevices coupled with automated image-based methods are now employed to analyze the instantaneous distribution and motion behaviors of microbial species in controlled environments at small temporal scales, mimicking, to some extent, macro conditions. Such technologies have so far been adopted for investigations mainly on individual species. Similar versatile approaches must now be developed for the characterization of multiple and complex interactions between a microbial community and its environment. Here, we provide a comprehensive step-by-step method for the characterization of species-specific behavior in a synthetic mixed microbial suspension in response to an environmental driver. By coupling accessible microfluidic devices with automated image analysis approaches, we evaluated the behavioral response of three morphologically different telluric species (Phytophthora parasitica, Vorticella microstoma, Enterobacter aerogenes) to a potassium gradient driver. Using the TrackMate plug-in algorithm, we performed morphometric and then motion analyses to characterize the response of each microbial species to the driver. Such an approach enabled to confirm the different morphological features of the three species and simultaneously characterize their specific motion in reaction to the driver and their co-interaction dynamics. By increasing the complexity of suspensions, this approach could be integrated in a framework for phenotypic analysis in microbial ecology research, helping to characterize how key drivers influence microbiota assembly at microbiota host-environment interfaces.

Coordination of two opposite flagella allows high-speed swimming and active turning of individual zoospores.

Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when their biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its gait from sinusoidal waves to power and recovery strokes, similar to Chlamydomonas's breaststroke, to reorient its body to a new direction. Our study is a fundamental step toward a better understanding of the spreading of plant pathogens' motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated 'run-and-tumble' motility class exhibited by peritrichous bacteria.

Microorganisms of the Phytophthora genus are serious agricultural pests. They cause diseases in many crops, including potato, onion, tomato, tobacco, cotton, peppers, and citrus. These diseases cause billions of dollars in losses each year. Learning more about how the tiny creatures disseminate and reach host plants could help scientists develop new ways to prevent such crop damage. The spore cells of Phytophthora, also known as zoospores, have two appendages called flagella on their bodies. A tinsel-shaped flagellum is near the front of the creature and a long smooth filament-like flagellum is near the posterior. Zoospores use their flagella to swim at high speeds through liquid toward potential plant hosts. Their complex swimming patterns change in response to different physical, chemical, and electrical signals in the environment. But exactly how they use their flagella to generate these movements is not clear. Tran et al. reveal new details about zoospore locomotion. In the experiments, Tran et al. recorded the movements of zoospores in a tiny 'swimming pool' of fluid on top of a glass slide and analyzed the movements using statistical and mathematical models. The results uncovered coordinated actions of the flagella when zoospores swim in a straight line and when they turn. The tinsel-like front flagellum provides most of the force that propels the zoospore forward. To do this, it beats with an undulating wave pattern. It shifts the beating to a breast-stroke pattern to change direction. The posterior flagellum provides a smaller forward thrust and temporarily pauses during turns. The study provides new details about zoospore's movements that may help scientists develop new strategies to control these pests. It also offers more information about how flagella coordinate their actions to switch speeds or change directions that may be of interest to other scientists studying organisms that use flagella to move.

Transcriptomic and Ultrastructural Signatures of K+-Induced Aggregation in Phytophthora parasitica Zoospores.

Most pathogenic oomycetes of the genus Phytophthora spread in water films as flagellated zoospores. Zoospores perceive and produce signals attracting other zoospores, resulting in autoaggregation in vitro or biofilm formation on plant surface. The mechanisms underlying intercellular communication and consequent attraction, adhesion and aggregation are largely unknown. In Phytophthora parasitica, the perception of a K+ gradient induces coordinated motion and aggregation. To define cellular and molecular events associated with oomycete aggregation, we combined transcriptomic and ultrastructural analyses. Results indicate involvement of electroception in K+ sensing. They establish that the transcriptome repertoire required for swimming and aggregation is already fully functional at zoospore release. At the time points analyzed, aggregates are mainly constituted of zoospores. They produce vesicular and fibrillary material discharged at cell-to-cell contacts. Consistently, the signature of transcriptome dynamics during transition to aggregates is an upregulation of genes potentially related to vesicular trafficking. Moreover, transcriptomic and functional analyses show a strong enhancement of carbonic anhydrase activity, indicating that pH homeostasis may contribute to aggregation by acting on both zoospore movement and adhesion. This study poses the molecular and cellular bases of aggregative behavior within oomycetes and expands the current knowledge of ion perception-mediated dissemination of propagules in the rhizosphere.

Surface tension propulsion of fungal spores.

Most basidiomycete fungi actively eject their spores. The process begins with the condensation of a water droplet at the base of the spore. The fusion of the droplet onto the spore creates a momentum that propels the spore forward. The use of surface tension for spore ejection offers a new paradigm to perform work at small length scales. However, this mechanism of force generation remains poorly understood. To elucidate how fungal spores make effective use of surface tension, we performed a detailed mechanical analysis of the three stages of spore ejection: the transfer of energy from the drop to the spore, the work of fracture required to release the spore from its supporting structure and the kinetic energy of the spore after ejection. High-speed video imaging of spore ejection in Auricularia auricula and Sporobolomyces yeasts revealed that drop coalescence takes place over a short distance ( approximately 5 microm) and energy transfer is completed in less than 4 mus. Based on these observations, we developed an explicit relation for the conversion of surface energy into kinetic energy during the coalescence process. The relation was validated with a simple artificial system and shown to predict the initial spore velocity accurately (predicted velocity: 1.2 m s(-1); observed velocity: 0.8 m s(-1) for A. auricula). Using calibrated microcantilevers, we also demonstrate that the work required to detach the spore from the supporting sterigma represents only a small fraction of the total energy available for spore ejection. Finally, our observations of this unique discharge mechanism reveal a surprising similarity with the mechanics of jumping in animals.

Guidance of zoospores by potassium gradient sensing mediates aggregation.

The biflagellate zoospores of some phytopathogenic Phytophthora species spontaneously aggregate within minutes in suspension. We show here that Phytophthora parasitica zoospores can form aggregates in response to a K+ gradient with a particular geometric arrangement. Using time-lapse live imaging in macro- and microfluidic devices, we defined (i) spatio-temporal and concentration-scale changes in the gradient, correlated with (ii) the cell distribution and (iii) the metrics of zoospore motion (velocity, trajectory). In droplets, we found that K+-induced aggregates resulted from a single biphasic temporal sequence involving negative chemotaxis followed by bioconvection over a K+ gradient concentration scale [0-17 mM]. Each K+-sensing cell moved into a region in which potassium concentration is below the threshold range of 1-4 mM, resulting in swarming. Once a critical population density had been achieved, the zoospores formed a plume that migrated downward, with fluid advection in its wake and aggregate formation on the support surface. In the microfluidic device, the density of zoospores escaping potassium was similar to that achieved in droplets. We discuss possible sources of K+ gradients in the natural environment (zoospore population, microbiota, plant roots, soil particles), and implications for the events preceding inoculum formation on host plants.

Bubble dynamics inside an outgassing hydrogel confined in a Hele-Shaw cell.

We report an experimental study of bubble dynamics in a non-Newtonian fluid subjected to a pressure decrease. The fluid is a hydrogel, composed of water and a synthetic clay, prepared and sandwiched between two glass plates in a Hele-Shaw geometry. The rheological properties of the material can be tuned by the clay concentration. As the imposed pressure decreases, the gas initially dissolved in the hydrogel triggers bubble formation. Different stages of the process are observed: bubble nucleation, growth, interaction, and creation of domains by bubble contact or coalescence. Initially bubble behave independently. They are trapped and advected by the mean deformation of the hydrogel, and the bubble growth is mainly driven by the diffusion of the dissolved gas through the hydrogel and its outgassing at the reactive-advected hydrogel-bubble interface. In this regime, the rheology of the fluid does not play a significant role on the bubble growth. A model is proposed and gives a simple scaling that relates the bubble growth rate and the imposed pressure. Carbon dioxide is shown to be the gas at play, and the hydrogel is degassing at the millimeter scale as a water solution does at a smaller scale. Later, bubbles are not independent anymore. The growth rate decreases, and the morphology becomes more anisotropic as bubbles interact because they are separated by a distance smaller than the individual stress field extension. Our measurements show that the interaction distance scales with the bubbles' size.

Stability of the hydrophilic and superhydrophobic properties of oxygen plasma-treated poly(tetrafluoroethylene) surfaces.

Poly(tetrafluoroethylene) (PTFE) materials were exposed to low and high-energy oxygen plasma, and the stability of the materials' surface was evaluated using contact angle, surface roughness, and surface chemistry characterizations. Lower-energy oxygen plasma treatments exhibited hydrophilic behavior with contact angles as low as 87°, and the higher-energy oxygen plasma treatments exhibited superhydrophobic behavior with contact angles as high as 151°. The wettability of all the treated samples as stored in air and in water was found to be stable in time as evidenced by the statistically insignificant differences in the advancing, receding, and hysteresis contact angles. Low contact angle hysteresis (θH<5°) and low sliding angle (α≈4°) were exhibited by the superhydrophobic surface. The surface morphology was found to be responsible for the changes in the wettability of the PTFE samples since (1) there was an increase in the surface rms roughness as the plasma discharge energy was increased, and (2) there were no significant changes in the observed group frequencies of the FT-IR spectra of the treated PTFE from the untreated PTFE.

Propagation of acoustic waves in a one-dimensional array of noncohesive cylinders.

By means of a photoelastic method, we access the visualization of acoustic waves propagating in a one-dimensional array of noncohesive cylinders. As pointed by Nesterenko in the case of spherical grains [V. F. Nesterenko, J. Appl. Mech. Tech. Phys. 24, p. 567 (1983)], the nonlinearity of the contact law between the grains induces a dependence of the wave velocity both on its amplitude and on the confinement force. Our experimental method allows one to access the evolution in time of the internal state of stress of individual grains with excellent accuracy. We show that the velocity of the sound presents two regimes as a function of the confining force. For low forces, the dependence is strongly nonlinear, while it weakens for higher forces. By means of the direct visualization of the contact zone, we show that both micro- and macroscale imperfections of the surface of contact explain the low forces behavior. We test the consistency of our experimental findings results with both the theoretical expectations and with the experimental determination of the force-displacement dependence. We show, moreover, that the main damping process originates in solid friction.

Ratchetlike motion of a shaken drop.

We study the motion of a drop lying on a plate simultaneously submitted to horizontal and vertical harmonic vibrations. The two driving vibrations are adjusted to the same frequency, and, according to their relative amplitude and phase difference DeltaPhi, the drop experiences a controlled directed motion with a tunable velocity. We present a simple model putting in evidence the underlying mechanism leading to this ratchetlike motion of the drop. Our model includes the particular case DeltaPhi=pi corresponding to the climbing of a drop on a vertically vibrated inclined substrate, as recently observed by Brunet et al. [Phys. Rev. Lett. 99, 144501 (2007)10.1103/PhysRevLett.99.144501]. This study gives insights in the fundamental issue of wetting dynamics and offers new possibilities of controlled motion in droplet microfluidics applications.