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Yeasts are unicellular fungi, which can be useful (bread, beer) or harmful to humans. Candida albicans is an opportunistic human pathogen, which can cause superficial as well as systemic infections, often life-threatening. One of the virulence factors of this organism is its ability to transit between an ovoid form and a filamentous form (hypha), capable of penetrating cellular tissues. During my thesis I studied the growth of C. albicans hyphae (diameter approximately 2 microns) in PDMS elastomers of various rigidity. I have observed that these hyphae are able to penetrate such environments with rigidities comparable to human tissues. My work was held into two parts.On one hand, I examined the penetration in cross-linked PDMS of a macroscopic one-millimeter diameter probe to better understand the mechanisms and forces involved, by analogy with the perforation of hyphae of microscopic size. I measured the force-strain relationships up to the point where the probe protrudes from the PDMS sample, which is a completely original aspect compared to the state of the art. I was able to show how the critical breaking force varied, but also the forces resistant to penetration up to the exit of the probe and to quantify the effects of the rigidity of the PMDS, but also of the speed.On the other hand, I worked with wild and mutated C. albicans strains. I developed and measured their growth in microfabricated PMDS membrane with chambers using real-time imaging. I first did a lot of work designing these membranes, most of the observations required a short working distance, which added a strong challenge on the manufacture of these membranes. The penetration of rigid media imposes on the filaments resistive stresses to their growth, as close as possible to the real situation. This makes the proposed approach an original one. Thanks to the results of the first part, we were able to determine the turgor pressure displayed in the hyphae using a recent growth model.; Les levures sont des champignons unicellulaires, qui peuvent être utiles (pain, bière) ou délétères pour l’homme. Candida albicans est une levure pathogène opportuniste de l’Homme, pouvant provoquer des infections superficielles mais aussi systémiques, pour lesquelles le pronostic vital est souvent engagé. Un des facteurs de virulence de cet organisme est sa capacité à transiter entre une forme ovoïde et une forme filamenteuse (hyphe), capable de pénétrer les tissus cellulaires. J’ai étudié pendant ma thèse la croissance d’hyphes de C. albicans (diamètre 2 microns environ) dans des élastomères de PDMS plus ou moins rigides. J’ai pu observer que ces hyphes sont capables de pénétrer de tels milieux aux rigidités comparables aux tissus humains. Mon travail s’est développé en deux parties. Dans une première partie, j’ai étudié la pénétration dans du PDMS réticulé d’une tige macroscopique millimétrique pour mieux comprendre les mécanismes et forces en jeu, par analogie avec la perforation des hyphes de taille microscopique. J’ai mesuré les relations forces-déformation jusqu’au point où la tige ressort de l’échantillon de PDMS, ce qui constitue un aspect complétement original par rapport à l’état de l’art. J’ai pu montrer comment variait la force critique de rupture, mais aussi les forces résistantes à la pénétration jusqu’à la sortie de la tige, et quantifier les effets de la rigidité du PMDS, mais aussi de la vitesse. Dans une deuxième partie, j’ai manipulé des souches de C. albicans sauvage et mutées. J’ai observé et mesuré leur croissance dans des dispositifs en PMDS microfabriqués à puits par imagerie en temps réel. J’ai d’abord réalisé un travail important de conception de ces dispositifs, sachant que la plupart des observations nécessitaient une distance de travail faible, ajoutant une contrainte forte sur la fabrication de ces dispositifs. La pénétration de milieux de rigidité et contrainte de rupture contrôlables imposent aux filaments des forces résistantes à leur croissance, au plus proche de la situation réelle, qui fait de l’approche proposée une démarche originale. Grâce aux résultats de la première partie, nous avons pu déterminer la pression de turgescence générée dans les hyphes en utilisant un modèle récent de croissance. more...

Additional file 1: Figure S1. Strain versus stress dependence of PDMS. Analyses carried out using a Viscoanalyzer, with oscillation at 10 Hz of PDMS at cross-linker ratio of 40:1. Figure S2. Invasive growth and penetration into adjacent chamber in PDMS of different stiffness. DIC time-lapse experiments at indicated PDMS:cross-linker ratio and measured stiffness (Young’s modulus). The adjacent chamber is highlighted with a dotted yellow line and deformation of this chamber lasted ~40 min with 40:1 PDMS ratio and 80-90 min for the two stiffer PDMS substrates. Figure S3. The shape of filament tip is not substantially altered during invasive growth in PDMS. A) Radius of curvature over time is constant in surface and invasively growing cells. Radius of curvature with an arc of ± 90° or ± 45° at the filament tip. B) Shape of filament tip of surface growing cells over time. Cells were grown on PDMS (30:1; 250 kPa) and 31 × 5 min GFP sum projections were analyzed. Radius of curvature with ± 45° by indicated open lines and ± 90° indicated by solid lines. Figure S4. Cells confined within a stiff PDMS chamber have reduced filament extension rates. A) Constricted growth within a PDMS chamber. Typical time-lapse experiment using 160 kPa PDMS, with DIC images every 5 min shown. B) Filament extension rate within a stiff chamber is not linear. Filament length was determined from images every 5 min for ~ 2 h and GFP sum projections (n = 9 cells). C) Filament extension rate is substantially reduced as chamber fills up. Initial (filament length 10-20 μm) and final (filament length > 20 μm) extension rates were determined from fits to 6 × 5 min GFP sum projections. (colors represent individual cells). Bars indicate SD and **** p < 0.0001. Figure S5. Distribution of active Cdc42 is not altered during invasive growth. A) Schematic indicating fluorescence signal over the filament long axis. Quantitation of slope of Gaussian farthest from tip in red (Max Slope, in relative units), distance maximum signal to tip (xmax in μm), and half width half max of the Gaussian farthest from tip in red (xSpread-xmax), i.e. the signal spread (Spread in μm). Signal is denoted by I and distance from tip by x. B) Distribution of active Cdc42 during surface and invasive filamentous growth. Experiment described in Figure 11a and 11b with the mean signal for each cell (colors represents individual cells), normalized to the mean signal for tip Cdc42•GTP in surface growing cells. Bars indicate SD. C) Distribution of active Cdc42 is not altered upon invasive growth. Relative maximum slope (left), distance from maximum signal to the tip (middle) and spread of signal (right) determined from 6-8 cells, using tailor-made Matlab program. Bars indicate SD; surface and invasive cells were not significantly different. D) Apical and subapical active Cdc42 signals are stable over time. Relative signals from apical and subapical region of sum projections, normalized to maximum invasive subapical signal. more...

Additional file 4: Table S1. Strains used in the study [61, 62]. Table S2. Oligonucleotides used in the study. Table S3. Synthesized DNA used in the study.