Aurélien Dumètre
email: aurelien.dumetre@univ-amu.fr

Jitender P Dubey
email: jitender.dubey@ars.usda.gov

David J P Ferguson

Pierre Bongrand

Nadine Azas

Pierre-Henri Puech
email: pierre-henri.puech@inserm.fr

Mechanics of the Toxoplasma gondii oocyst wall

The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from harsh environmental conditions until their ingestion by the host. None of the common disinfectants are effective in killing the parasite, since the oocyst wall acts as a primary barrier to physical and chemical attacks.

Here, we address the structure and chemistry of the wall of the T. gondii oocyst by combining wall surface treatments, fluorescence imaging, electron microscopy and measurements of its mechanical characteristics by using Atomic Force Microscopy (AFM). Elasticity and indentation measurements indicated that the oocyst wall resembles common plastic materials, based on the Young moduli, E, evaluated by AFM. Our study demonstrates that the inner layer is as robust as the bilayered wall itself. Besides wall mechanics, our results suggest important differences regarding the non specific adhesive properties of each layer. All together, these findings suggest a key biological role for the oocyst wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or after exposure to disinfectants, and therefore their potential infectivity to humans and animals.

Introduction

Resistance to physical and chemical degradation is essential for the perpetuation of the life cycle of environmentally exposed microbial pathogens. In the coccidian parasite Toxoplasma gondii, this function is served by the oocyst, the only stage of the parasite structurally equipped to survive in harsh environments [START_REF] Dubey | Toxoplasmosis of animals and humans[END_REF]. Oocyst-related infections in humans and other warm-blooded animals worldwide have been increasingly reported as more prevalent and severe than previously thought [START_REF] Bahia-Oliveira | Highly endemic, waterborne toxoplasmosis in north Rio de Janeiro state, Brazil[END_REF][START_REF] Moura | Waterborne toxoplasmosis, Brazil, from field to gene[END_REF][START_REF] Jones | Waterborne toxoplasmosis-recent developments[END_REF][START_REF] Boyer | Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America[END_REF][START_REF] Dattoli | Oocyst ingestion as an important transmission route of Toxoplasma gondii in Brazilian urban children[END_REF]. Infections lead to possible deleterious ocular and neurological complications, and even death [START_REF] Robert-Gangneux | Epidemiology of and diagnostic strategies for toxoplasmosis[END_REF]. In this context, a global effort has emerged to decipher the basic structures [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF]9) and biological processes of the oocyst [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF][START_REF] Fritz | Transcriptomic analysis of Toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF][START_REF] Possenti | Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation[END_REF] that allow the parasite to survive different environmental conditions and disinfectants [START_REF] Dumètre | Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxoplasma gondii oocysts[END_REF][START_REF] Wainwright | Chemical inactivation of Toxoplasma gondii oocysts in water[END_REF][START_REF] Wainwright | Physical inactivation of Toxoplasma gondii oocysts in water[END_REF][START_REF] Villegas | Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers[END_REF](18).

The oocyst is the result of sexual multiplication of T. gondii in the intestinal epithelium of cats [START_REF] Dubey | Cyst-induced toxoplasmosis in cats[END_REF][START_REF] Ferguson | Ultrastructural study of early stages of asexual multiplication and microgametogony of Toxoplasma gondii in the small intestine of the cat[END_REF][START_REF] Ferguson | The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii[END_REF]. A few days post-infection, unsporulated (uninfective) spheroid oocysts [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF] x 12 µm) are excreted in cat feces and become rapidly infective following aerobic sporulation [START_REF] Dubey | The Toxoplasma gondii oocyst from cat feces[END_REF]. Sporulation results in different subpopulations of maturing oocysts during the first few days [START_REF] Dubey | The Toxoplasma gondii oocyst from cat feces[END_REF]: unsporulated (NS), 'sporoblast-staged' (SB) and fully sporulated (SP) oocysts [START_REF] Fritz | Transcriptomic analysis of Toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts[END_REF][START_REF] Dubey | The Toxoplasma gondii oocyst from cat feces[END_REF]. SP oocysts are ovoid, measure 11 x 13 µm in size and have two sporocysts (6 x 8 µm), each containing four infective banana-shaped sporozoites (2 x 6-8 µm) (SI Appendix, Fig. S1) [START_REF] Ferguson | Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. III. Formation of the sporozoites within the sporocysts[END_REF][START_REF] Speer | Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii[END_REF].

Toxoplasma gondii oocysts are highly resistant to environmental influences and this resistance to various physical and chemical stressors, including disinfectants such as UV, ozone, and chlorine-based products, is attributed to the oocyst wall [START_REF] Jones | Waterborne toxoplasmosis-recent developments[END_REF][START_REF] Dumètre | How to detect Toxoplasma gondii oocysts in environmental samples?[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF]. In contrast, oocysts are rapidly inactivated following exposure to temperatures above 60°C 3 5 6 for few minutes (27). The oocyst wall is bilayered with the outer layer being thinner than the inner layer [START_REF] Speer | Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii[END_REF]. The layers are not tightly bound to each other since the outer layer can be stripped off easily using sodium hypochlorite [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF][START_REF] Dubey | The Toxoplasma gondii oocyst from cat feces[END_REF], and the present study).

The oocyst wall is made of more than 90% proteins with several structural cysteine-and tyrosine-rich proteins having been identified in the outer layer only [START_REF] Possenti | Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation[END_REF] or in whole oocyst wall fractions [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF]. How these different proteins are processed and/or packed to form the oocyst wall is still not clear [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF]. Current models support a strong contribution of protein-tyrosine cross-linking in the formation and hardening of the oocyst wall in Toxoplasma-related coccidia [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF][START_REF] Belli | Roles of tyrosinerich precursor glycoproteins and dityrosine-and 3,4-dihydroxyphenylalaninemediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima[END_REF] and results in the development of its typical blue autofluorescence (AF) under UV excitation [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF][START_REF] Belli | Roles of tyrosinerich precursor glycoproteins and dityrosine-and 3,4-dihydroxyphenylalaninemediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima[END_REF][START_REF] Lindquist | Autofluorescence of Toxoplasma gondii and related coccidian oocysts[END_REF] (SI Appendix, Fig. S1B).

This complex polymeric organization also suggests an important robustness of the T. gondii oocyst wall in terms of mechanics [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF]. Thus, measuring mechanical properties of the T. gondii oocyst wall appears to be relevant to addressing the respective roles of structure and chemistry of each layer of the oocyst wall in the overall resistance of the oocyst to various physical and chemical agents [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF].

Atomic Force Microscopy (AFM) is a rather new technique in biology that fits perfectly for this purpose [START_REF] Franz | Atomic Force Microscopy: A versatile tool for studying cell morphology, adhesion and mechanics[END_REF]. The AFM provides valuable information regarding the surface topography and/or allows force measurements in physiological media, with fixed or unfixed samples, from proteins to cells [START_REF] Muller | Force nanoscopy of living cells[END_REF][START_REF] Muller | New frontiers in atomic force microscopy: analyzing interactions from single-molecules to cells[END_REF][START_REF] Müller | Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology[END_REF]. AFM uses a finger-like tip, at the extremity of a very soft cantilever [START_REF] Franz | Atomic Force Microscopy: A versatile tool for studying cell morphology, adhesion and mechanics[END_REF]. This tip can be used to: (i) delineate the surface (imaging mode, [START_REF] Friedrichs | Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM[END_REF][START_REF] Muller | AFM: a nanotool in membrane biology[END_REF][START_REF] Müller | Single-molecule studies of membrane proteins[END_REF]), (ii) indent the objects surface by pressing on them allowing measurement of their mechanical properties (force mode, mechanics, (37-40)) or (iii) probe the adhesion of surface molecules when decorated with suitable haptens and 4 7 pulling the lever off the surface until all built bridges are broken. This allows direct quantifying of the force that these bridges can sustain (force mode, adhesion). For modes (ii) and (iii), the AFM cantilever is held at a given x,y above the sample (a surface or a cell) and ramped in z-direction. Measuring cantilever deflection as a function of piezo position produces force-extension curves (FC) [START_REF] Franz | Atomic Force Microscopy: A versatile tool for studying cell morphology, adhesion and mechanics[END_REF].

Using the AFM tip as a microindentor and using the part of the FC where the tip is pressed on the surface, one can gain information regarding the local elastic properties as measured as a Young's modulus, E, using a Hertz model for elastic indentation. E moduli measured for different eukaryotic cell types vary greatly from cell type to cell type and usually ranges from 1 kPa to several 100 kPa (37,[START_REF] Radmacher | Studying the mechanics of cellular processes by atomic force microscopy[END_REF]. Using the part of the FC where the tip is retracted from the surface, adhesion force measurements can be performed and have been employed in cell biology, from single molecules measurements to cell/cell measurements [START_REF] Muller | Force nanoscopy of living cells[END_REF][START_REF] Puech | A new technical approach to quantify cell-cell adhesion forces by AFM[END_REF][START_REF] Fierro | BCR/ABL expression of myeloid progenitors increases beta1integrin mediated adhesion to stromal cells[END_REF][START_REF] Taubenberger | Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy[END_REF][START_REF] Franz | Studying integrinmediated cell adhesion at the single-molecule level using AFM force spectroscopy[END_REF][START_REF] Puech | Force measurements of TCR/pMHC recognition at T cell surface[END_REF][START_REF] Puech | Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy[END_REF](47). The sensitivity of force determination is usually limited by the thermal noise of the system and the properties of the chosen cantilever [START_REF] Franz | Atomic Force Microscopy: A versatile tool for studying cell morphology, adhesion and mechanics[END_REF].

Here, we investigate using AFM the mechanics of the wall of T. gondii oocysts submitted to physical (heat inactivation) and chemical (bleach exposure) treatments separate or in combination in order to evaluate the contribution of each layer in the overall mechanics of the oocyst wall. Our results present a simple way to gently but firmly immobilize oocysts on surfaces to image them using fluorescence microscopy or test their mechanics using AFM under moderate forces. Our findings may be correlated to structural modifications of the oocyst wall and suggest a key biological role of the wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or exposed to disinfectants, therefore affecting potential infectivity to humans and animals.

Results

Microscopic Characteristics of T. gondii Oocysts Following Sporulation in Water.

In order to evaluate the basic mechanics of the T. gondii oocyst wall, we limited the use of chemicals to avoid any modification of the wall structure due to handling or storage conditions. For this, oocysts were sporulated in water rather than in 2% aqueous sulfuric acid solution which is commonly used for oocyst sporulation and subsequent storage [START_REF] Fritz | Transcriptomic analysis of Toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts[END_REF][START_REF] Dubey | The Toxoplasma gondii oocyst from cat feces[END_REF][START_REF] Ferguson | Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. I. Development of the zygote and formation of the sporoblasts[END_REF]. After a 5-day sporulation process, the oocyst suspension contained 18.6 ± 2.7 % of NS, 18.3 ± 6.4 % of SB and 63.0 ± 5.9 % of SP oocysts (SI Appendix, Fig. S1). All these different oocyst subpopulations exhibited the same typical autofluorescence (AF) under UV excitation (SI Appendix, Fig. S1B). Careful observation, before and during AFM experiments, was a key step to the exact categorization of the objects.

Characterization of the Bleach Effects on T. gondii Walls by Electron Microscopy.

Electron microscopy confirmed that control (H 2 O-conserved) oocysts retained their typical double-layered wall (observed thickness ~50 nm, Fig. 1A,B). In contrast, the outer layer (observed thickness ~18-20 nm) was absent in bleach-treated oocysts with only the inner layer (observed thickness ~30 nm) remaining with, in certain cases, slight remnants of the outer layer persisting (Fig. 1C). The oocyst wall thicknesses that were observed here are consistent with but in the lower end of the reported values of such structures reported in literature (up to 100 nm for the bilayered wall) [START_REF] Speer | Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii[END_REF]. It is interesting to note that the bilayered structure of the T. gondii sporocyst wall was maintained following bleach treatment of the oocyst wall (Fig. 1D,E).

11

Characterization of T. gondii Walls by Fluorescence Microscopy.

The properties of the wall of NS, SB and SP oocysts were first assessed microscopically by analyzing their reactivity to the monoclonal antibody (mAb) 4B6, which is specific to the inner layer of the oocyst wall [START_REF] Dumètre | Detection of Toxoplasma gondii in water by an immunomagnetic separation method targeting the sporocysts[END_REF]. In order to induce structural modifications of the bilayered oocyst wall, the parasites were treated by bleach to remove the outer layer and/or heated at 80°C.

In contrast to bleach treatment, heating oocysts at 80°C efficiently kills the sporozoites, however, the effects on the wall structure remain largely unknown. Oocysts exposed only to water during their maturation and storage served as controls. The percentages of oocysts at different maturing stages exposing partially or totally their inner wall following these different surface treatments are shown in Fig. 2 (Fig. 2). A mixture of unstained to totally mAb 4B6-stained oocysts were observed irrespective of the oocyst developmental stage (SI Appendix, Fig. S2A). Fluorescent staining appeared randomly distributed at the oocyst surface, and ranged from almost continuous staining of the entire surface (SI Appendix, Fig. S2A, case 1) to very discrete patches (SI Appendix, Fig. S2A, case 2). In these conditions, 4B6 staining of the inner wall layer appeared to result from the infiltration of the antibody through cracks in the oocyst wall rather than the exposure of the outer aspect of the inner layer of the oocyst wall. This hypothesis was further supported by the fact that ~25-30% of H 2 O-exposed oocysts were permeable to FITC in solution, again indicating possible openings in the 7 13 oocyst wall (SI Appendix, Fig. S3). Irrespective of their 4B6 pattern, SB and SP oocysts were more autofluorescent than NS oocysts (SI Appendix, Fig. S2A and Fig. S4).

Heating oocysts at 80°C for 10 min led to a significant reduction of the percentage of 4B6-positive oocysts at all maturation stages (0.6-3.1%) (Fig. 2). The few positive oocysts observed were very faintly stained in localized areas of the oocyst wall (SI Appendix, Fig. S2B). In these experimental conditions, heating did not appear to alter significantly the microscopic structure or internal content of NS, SB and SP oocysts or the AF of the oocyst wall (SI Appendix, Fig. S2B compared to Fig. S2A).

In contrast, there was a significant increase in mAb 4B6 staining of oocysts bleached with 3% bleach solution for 30 min (final mAb 4B6-labelled oocysts from 48.3 to 80.7% depending on the maturing stage) compared to control or heated oocysts (Fig. 2). In these experimental conditions, the antibody stained, at least partially, the inner layer of NS, SB and SP oocyst wall usually with a strong intensity (SI Appendix, Fig. S2C) indicating that modifications of the wall structure increase the access of mAb 4B6 to the outer aspect of the inner layer of the wall, but not its infiltration through it since only ~25-30% of bleach-exposed oocysts were again permeable to FITC due to possible fractures in their wall (SI Appendix, Fig. S3). We also observed that several bleach-treated oocysts displayed a reduced AF pattern of the oocyst wall compared to the control oocysts (SI Appendix, Fig. S4, suggesting possible differences in the biochemical content of the oocyst wall between untreated and bleach-treated oocysts.

When bleached oocysts were subsequently heated at 80°C, the percentages of mAb 4B6positive parasites were similar to that observed with bleaching alone (40.1-74.3%) (Fig. 2). Microscopically, these oocysts had similar 4B6 and AF patterns compared to bleached-only oocysts, irrespective of the stage of maturation (SI Appendix, Fig. S2D compared to Fig. S2C). However, a few mAb 4B6-positive oocysts still had a very discrete wall AF (SI Appendix, Fig. S2D, case 5).

Attachment of the Oocysts for AFM Experiments. The mechanical properties of the wall of H 2 O-stored, bleach-and/or heat-treated oocysts at different maturing stages were then evaluated using AFM. For this, oocysts were first allowed to adhere onto Poly-L-Lysine (PLL)-coated glass microscopic slides prior to being approached by the AFM tip (SI Appendix, Fig. S5A,B,C). We verified that the coating procedure on glass did not change significantly the initial observed proportions of the different subpopulations of the oocysts irrespective of their pretreatment (SI Appendix, Fig. S5B). This coating procedure was suitable for firmly attaching the oocysts onto the glass surface thus allowing repeated contacts between the AFM tip and each oocyst at a preset contact force of 1 nN. The fine positioning of the AFM tip on top of the substructures (i.e. sporocysts) does not largely affect the measurements (SI Appendix, Fig. S5D,E).

Elastic Properties of the Oocyst Wall. Following our immobilization protocol, repeated force curves were obtained for each adhered object (Fig. 3A,B). We observed that the indentation depth was <50 nm and typically ~ 20 nm (Fig. 3C,D) irrespective of the maturing oocyst stages or their pretreatment (SI Appendix, Table S1). This median indentation is of the same order of magnitude as the most outer layer of the oocyst wall (20 nm) and smaller than the thickness of the most inner layer (30 nm) as measured in EM micrographs. Following this, (i) in absence of bleach treatment, we concluded that 9 17 18 we measured the mechanics of, if not the outer layer alone, the bilayered structure of the wall, and (ii) following bleach treatment, we were able to access the mechanical properties solely of the inner layer of the wall.

Young moduli, E, obtained for NS, SB and SP H 2 O-stored oocysts were typically in the 10 6 -10 7 Pa range and were not significantly different from each other (Fig. 3E,F). Those elevated E values are similar to the ones reported for artificial polymeric capsules of comparable thicknesses [START_REF] Dubreuil | Elastic properties of polyelectrolyte capsules studied by atomic-force microscopy and RICM[END_REF]. The median E moduli showed no significant variation between the different maturing oocyst stages irrespective of the oocyst pretreatment (SI Appendix, Tables S2 andS4). Force relaxation experiments during contact of the tip on the oocyst and the superposition of trace and retrace parts of the force curves indicated that no viscous behavior could be identified in this range of stimulations (Fig. 3A,B). In addition, repetitions of contacts lead to rather unvarying indentation depths, with no apparent tendency of any plastic deformation of the oocyst wall for the investigated forces (Fig. 3C).

Pressing the oocysts at higher forces (30 or 120 nN) did not appear to strongly modify the mechanics of the oocyst wall (SI Appendix, Fig. S5F,G). However, in these conditions, we observed that oocysts were accidently removed from the slide because of the higher pressing force, making the AFM tip into a golf club and the oocyst the ball.

Adhesive Properties of the Oocyst Wall. In addition to indentation and elasticity measurements, the non-specific adhesive properties of the oocyst wall were examined.

Surface adhesion (Fig. 4) was measured as the force required to detach the AFM tip from the surface of the oocyst after indentation at 1 nN, with a fixed pulling speed of 1 µm/s. 10 19 20 We observed that at least 50% of the force curves showed some adhesion (Fig. 4A vs.

Fig 4B). The proportion of oocysts showing a detectable adhesion was observed to

depend on the developmental stage and on the treatment of the oocyst surface.

Temperature alone lowered the proportion of oocysts with adhesive surface properties while application of bleach increased it (Fig. 4C andD). Upon repeated pulling on the same object, adhesive forces did not show any marked tendency (Fig. 4E) indicating that materials coming from the wall did not pollute the tip. We observed that the strength of adhesion (i.e. the force required to fully detach the tip from the oocyst) was rather low (<100 pN) for control and temperature-treated oocysts but was significantly stronger when bleach was applied as a first treatment (Fig. 4F and SI Appendix, Table S3) suggesting important bleach-induced modifications of the wall. In conclusion, bleach treatment increases both the proportion of oocysts showing detectable adhesion and the overall strength of the adhesion while high temperature treatment alone did not affect adhesion force but reduces the proportion of oocysts showing significant adhesion.

Discussion

The T. gondii oocyst is a superstructure that protects the dormant but potentially infective sporozoites from many extreme conditions that would be deleterious for survival [START_REF] Speer | Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii[END_REF].

Facing the external environment, the oocyst wall acts as a primary barrier to physical and chemical attacks as long as its complex polymeric organization is perfectly maintained [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF]. Different, but complementary, approaches have been applied to investigate the structure and molecular basis of the oocyst wall resilience, such as electronic microscopy (9, 12, 24, 51) and proteomics studies [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF][START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF]. However, oocysts are 11 21 22 technically difficult to process for electron microscopy examination because of the impervious nature of the walls and proteomics usually require large numbers of highly purified oocysts, which are difficult to obtain since oocysts cannot be generated in vitro.

Here, we addressed the structure and chemistry of each oocyst wall layer by measuring their respective mechanical properties by combining wall treatments, fluorescence and electron microscopic observations, and AFM techniques on immobilized parasites.

In coccidian parasites such as T. gondii, the oocyst wall results from the particular arrangement of structural proteins through a sclerotization process involving both quinone tanning and protein dityrosine crosslinking and dehydration [START_REF] Mai | Oocyst wall formation and composition in coccidian parasites[END_REF][START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF]. This process probably takes place very early in the development of the oocyst wall, from the NS stage before it leaves the host [START_REF] Ferguson | The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii[END_REF], and is thought to lead to hardened structures, which excludes water-soluble molecules in order to form a complex polymeric covering capable of resisting extended physical and chemical-induced disorganization.

Interestingly, it has been recently claimed that the inner wall layer of Eimeria tenella oocysts possesses discrete pores of 5-250 nm (9). However, such structures in the T. gondii oocyst wall were not observed in the present or previous studies [START_REF] Belli | The coccidian oocyst: a tough nut to crack![END_REF].

The oocyst wall layers in T. gondii are assumed to differ in their thickness and molecular content, the inner one being thicker, less electron dense and more resistant to chemical degradation than the outer one [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF]. Using fluorescence imaging and electron microscopy combined with different treatments, we provide new insights on the structure and chemistry of the wall of T. gondii oocysts. Specific immuno-staining of the inner wall layer of H 2 O-exposed oocysts was infrequent and appeared to result from the infiltration of the antibody through focal openings of the oocyst wall rather than exposure of the 12 23 external surface of the inner layer. After heating, antibody staining was significantly decreased in control oocysts while it had little, if no, effect on bleach-treated oocysts. This would suggest that heating is not denaturing the antigen recognized by the antibody and probably results from heat-induced reticulation of poly-protein structures of the outer layer reducing penetration of the antibody. In contrast, bleach treatment clearly affected both the structure and chemistry of the oocyst wall, by removing the outer layer as seen in our EM micrographs and significantly affecting the oocyst wall AF. Consequently, most of the 4B6 staining observed in bleached oocysts could be linked to the exposure of the external surface of the inner layer of the oocyst wall.

Then, we investigated the mechanical properties of the double-layered oocyst wall, and then those specific to the inner layer after removing the outer layer by treatment with bleach. We observed high E moduli comparable to polymeric shells [START_REF] Dubreuil | Elastic properties of polyelectrolyte capsules studied by atomic-force microscopy and RICM[END_REF] with neither viscous nor plastic behaviors. The E moduli were not significantly different between the different maturing stages and treatments involving temperature and bleach (alone or in combination). Considering the small indentation and high E modulus, our results strongly support that the global stiffness of the bilayered oocyst wall is in the same order as that of the inner layer alone. Interestingly, we showed that the oocyst wall stiffness did not vary significantly following parasite incubation at 80°C, which was quite unexpected since heat-induced stresses usually result in increasing stiffness of polymeric multilayer microcapsules due to an increased shell thickness or reticulation [START_REF] Delcea | Mechanobiology: correlation between mechanical stability of microcapsules studied by AFM and impact of cell-induced stresses[END_REF]. The conservative hypothesis suggests that each wall layer retains its basic mechanical properties by maintaining to large extend its molecular architecture, even at this temperature.

25

Besides mechanical measurements, AFM has also permitted examination of the nonspecific adhesive properties of each layer of the wall. It was observed that the proportion of oocyst showing adhesive properties was lower as was the strength of the adhesion (i.e. overall force needed to fully detach the tip from the oocyst) in parasites retaining their typical double-layered wall structure, whereas there was a higher proportion of oocysts with adhesive properties and stronger adhesion was recorded in oocysts exposing solely the inner wall layer (SI Appendix, Fig. S6). This contrasting behavior might indicate the existence of important differences regarding the biochemical nature of the molecules and/or their arrangement from one wall layer to the other resulting from oocyst treatments modulating the structure of the wall. We speculate an increasing number of residual polypeptidic chains due to removal of (most of, if not all) the most outer layer of the wall (SI Appendix, Fig. S6). Such differences have also been hypothesized by others [START_REF] Fritz | Proteomic analysis of fractionated Toxoplasma oocysts reveals clues to their environmental resistance[END_REF], based on proteomics analyses of purified wall fractions of T. gondii oocysts exposed or not to bleach. It is not yet clear whether these different adhesive properties play a role in the oocyst fate. Recent studies have shown that the negative charges covering the surface of the oocyst wall prevent in most cases any aggregation of the parasites with other particles, thus allowing the parasites to disperse freely in fresh water [START_REF] Dumètre | Interaction forces drive the environmental transmission of pathogenic protozoa[END_REF]. Further investigations, in particular on molecule-specific adhesion and the effects of digestive enzymes found in the host's gut, are required to extend our study and refine the biological significance of the adhesive properties of the oocyst wall. In conclusion, our study demonstrates that the overall rigidity of the bilayered T. gondii oocyst wall is as high as common plastic materials and that the inner layer is as robust as the bilayered wall itself. These findings strongly suggest that the mechanical characteristics of the T. gondii oocyst wall sustain the survival of the enclosed sporozoites facing physical and chemical attacks outside the host. In particular, our results suggest that chlorine-based products used as surface disinfectants or for treating drinking water are ineffective for efficiently killing T. gondii oocysts because these compounds are not able to permeabilize or disrupt the oocyst wall. However, it is clear that these properties have to be circumvented following oocyst ingestion by the host, in order to safely deliver the sporozoites near the enterocytes. As chemicals are ineffective in breaking the oocyst wall, a supplementary physical stimulus (still to be determined) seems to be required to prime oocyst-related infections in humans and animals.

Materials and Methods

Oocyst Purification and Sporulation. Oocysts of the genotype II TgNmBr1strain of T. gondii [START_REF] Dubey | Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil[END_REF] were harvested from feces of cat 6-8 days after feeding infected mouse tissues to a T. gondii free cat (1), then purified by flotation, and allowed to sporulate at RT for 5 days. More details can be found in SI. Oocyst suspension was stored in distilled water at 4°C until used within 3 months. More details can be found in SI Appendix. Electron Microscopy. Samples of water maintained and bleached oocysts were mechanically ruptured prior to fixation in 2.5% glutaraldehyde in 0.1M phosphate buffer and processed for routine electron microscopy. In summary, the samples were post-fixed in osmium tetroxide, dehydrated in ethanol, treated with propylene oxide and embedded in Spurr's epoxy resin. Thin sections were cut and stained with uranyl acetate and lead citrate prior to examination in a Jeol 1200EX electron microscope.

Immunofluorescence Assay (IFA). The effects of bleach and heat treatments on the integrity of the oocyst wall were evaluated by IFA combined with the autofluorescent signal (AF). We labeled oocysts in suspension using a monoclonal antibody (IgM mAb 4B6), which was previously shown to react mainly with the inner layer of the oocyst wall [START_REF] Dumètre | Detection of Toxoplasma gondii in water by an immunomagnetic separation method targeting the sporocysts[END_REF][START_REF] Dumètre | Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall[END_REF]. More details can be found in SI Appendix. 16 [START_REF] Muller | Force nanoscopy of living cells[END_REF]

32

FITC Infiltration Assay. The permeability of the wall of oocysts exposed or not to bleach treatment was assessed by incubating oocysts with FITC at 0.5 mg/mL in PBS for 1 hr at room temperature. Oocysts were subsequently washed four times in PBS by gentle centrifugations at 5000 g for 5 min and examined for fluorescence of FITC bound to internal proteins (SI Appendix, Fig. S3).

Measurements of the Oocyst Wall Autofluorescence Intensity.

The effects of bleach treatment on the autofluorescence pattern of the oocyst wall were evaluated on AF gray scale images as described in SI Appendix, Fig. S4.

AFM Experiments

Oocyst Immobilization on Glass Coverslides. Clean glass coverslides were coated with poly-L-lysine (PLL) after activation using a residual air-based plasma After rinsing and mounting on an observation chamber, a diluted suspension of untreated, heat-inactivated, or bleach-treated oocysts was seeded onto the PLL treated zone and let to settle for 45min to 1 hr at RT before removal of non adherent objects. We observed that this procedure did not grossly affect the different sub-populations ratios as compared to the original suspension (SI Appendix, Fig. S5B). More details can be found in SI Appendix. AFM Measurements. A Nanowizard I (JPK Instruments, used in closed loop mode) sitting on an Axiovert 200 (Zeiss) equipped with 10x and 40x lenses (with an optional 1.6x lens) was used to measure the oocyst mechanics. The system was sitting on an active damping table (Halcyonics) to suppress mechanical noise. Blunt AFM levers (MLCT, Veeco, nominal spring constant 10 pN/nm) were used and calibrated in situ. The spring 17 33 34 constant (~ 15-18 pN/nm) was determined in situ using a built-in thermal calibration method, far from the glass surface to avoid any hydrodynamical bias due to the coupling with the substrate [START_REF] Butt | Calculation of thermal noise in atomic force microscopy[END_REF].

Using bright field, a given oocyst was chosen and a calibrated AFM cantilever was positioned on top of it (SI Appendix, Fig. S5C,D). We checked that the measurements were not affected by the fine positioning of the tip on top of the structures (e.g. over the two substructures of SP oocysts and between them, so only the mid position was used to quantify the structure's mechanics (SI Appendix, Fig. S5D,E). At least 10 force curves, with a preset contact force of 1 nN, a preset contact time of 0 sec, pressing/pulling speeds of 1 μm/sec and an acquisition frequency of 1024 Hz, were acquired per oocyst (Fig. 3A,B). Each force curve was evaluated by eye and processed on a PC using the built-in JPK IP software (JPK Instruments), resulting in 3 to more than 15 data points extracted for each structure. We first observed that the maximal indentation depth at 1 nN was in average ~ 20 nm (Fig. 3C,D), that is less than the thickness of the bilayered wall structure, so the model was accurate enough to allow us to extract the Young modulus of the structures, E, one per valid force curve. To quantify the mechanics, we used as a first approximation a Hertz model for contact to fit the pressing part of the force curves assuming a pyramidal tip of α=21° half angle (calculated from manufacturer data) and incompressibility of the material (ν=0.5) (37) (Fig. 3A,B). Since the indentation was <50 nm, we also tried the Hertz model for a spherical indentor of 25-50 nm radius and found little differences in the quality of the fit and subsequently calculated E moduli.

For adhesion measurements, we retrieved from the return/pulling part of the force curves 18 35 36 the maximum detachment force (Fig. 4B) and frequency of adhesion events (i.e. the ratio between the number of force curves having adhesion divided by the total number of force curves taken into account) (Fig 4C ,D). We then calculated median adhesion forces and plotted the entire force distribution as whisker plots. We did not observe any tendency of adhesion force versus upon repetitions of tests (Fig. 4E).

Experiments lasted maximum 2 hr at RT before changing the sample and the cantilever with occasional supplementations of water to counteract the evaporation. showing that for a given object no tendency can be observed upon repeated indentation.

Note that the value of 10 4 Pa used as the lower limit of the scale corresponds to the values recorded for hard eukaryotic cells as found in literature. (F) Young modulus as a function of oocyst subtype and treatment. In D and F, each point is the median value obtained for a single oocyst upon successive indentations. The line is the median of the subsequent distribution. No significant differences are observed between the conditions neither for indentation (D and SI Appendix, Tables S1,S4) nor for Young modulus (F and SI Appendix, Tables S2,S4). 31 [START_REF] Fritz | Transcriptomic analysis of Toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts[END_REF] 62 S3 andS4).

65 66

Supplement Information (SI) Appendix

Materials and Methods

Oocyst Purification and Sporulation. Oocysts of the genotype II TgNmBr1strain of T.

gondii [START_REF] Dubey | Toxoplasmosis of animals and humans[END_REF] were harvested from feces of cat 6-8 days after feeding infected mouse tissues to a T. gondii free cat. Oocysts were collected by flotation at 4°C from cat feces on a 1.15 specific gravity sucrose solution without phenol. Concentrated oocyst pellets were then resuspended in 5 mL of distilled water and sent with cold packs within 48 hours by FedEx from Beltsville, Maryland, USA to Marseille, France, for sporulation and further experiments. Upon arrival, oocysts were washed three times in distilled water, pelleted by centrifugation at 5000 g for 5 min at room temperature (RT, 20-22°C), then resuspended in 7 mL of distilled water, and transferred in a 100 mL small plastic container. Oocysts were allowed to sporulate at RT for 5 days under adequate aeration and gentle continuous shaking. Sporulation progress was monitored daily by examining a fraction of the suspension by using bright field and UV microscopy as described elsewhere [START_REF] Bahia-Oliveira | Highly endemic, waterborne toxoplasmosis in north Rio de Janeiro state, Brazil[END_REF][START_REF] Moura | Waterborne toxoplasmosis, Brazil, from field to gene[END_REF].

Oocyst suspension was stored in distilled water at 4°C until used within 3 months.

Immunofluorescence Assay (IFA).

The effects of bleach and heat treatments on the integrity of the oocyst wall were evaluated by IFA combined with the autofluorescent signal (AF). We used a monoclonal antibody (IgM mAb 4B6), which was previously shown to react mainly with the inner layer of the oocyst wall [START_REF] Jones | Waterborne toxoplasmosis-recent developments[END_REF][START_REF] Boyer | Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America[END_REF]. This labeling was performed on oocysts in suspension rather than on air-dried parasites because drying on slides frequently induces the opening of the oocyst wall, which invariably gives to the mAb 4B6 an access to the inner layer [START_REF] Jones | Waterborne toxoplasmosis-recent developments[END_REF][START_REF] Boyer | Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America[END_REF].

Briefly, untreated (H 2 O-stored), heat-inactivated and bleach-treated oocysts were allowed to react with mAb 4B6 diluted at 1:100 in PBS for 30 min at 37°C. Oocysts were subsequently washed three times in PBS by gentle centrifugations at 5000 g for 5 min prior to incubation with a goat fluorescein-isothiocyanate (FITC) conjugate anti-mouse IgM+IgG (50-011, Argene, France) diluted to 1:100 in PBS. After that, parasites were washed again three times in PBS using the same protocol. Samples are then mounted and 35 69 70 examined on a BX51 microscope (Olympus, France) equipped with suitable epifluorescence filters for FITC and UV autofluorescence (AF) and 40x lens. Bright field, FITC and AF images were acquired using the fluorescence imaging system Cell A (Olympus, France) and quantified using ImageJ 1.46m. The normally blue AF signal (Fig. S1B) was placed in the red channel and the FITC in the green channel for more convenient merging when performing fluorescence colocalization (Fig. S2).

Oocyst Immobilization on Glass Coverslides. Coverslides were cleaned using a 10% v/v Helmanex (Helma) solution in water in an ultrasonic bath for 30 min at 60°C, subsequently separately rinsed using alternating baths of ethanol and MQ water (three of each). Then, a supplementary cleaning in MQ water in an ultrasonic bath for 30 min at 60°C was performed before a last rinsing step with MQ water prior to drying under an air flow. Coverslides were stored away from humidity and dust for up to two weeks before use. For coating with poly-L-lysine (PLL), clean coverslides were activated for 1 min using a residual air-based plasma cleaner and a PDMS stamp with a circular 8 mm in diameter hole was stuck on them. 100 μL of 0.01% PLL in water was added and incubated for 45 min to 1 hr at RT. Three rinsing with PBS were performed before gluing a plastic ring with vacuum grease after removal of the PDMS stamp. The resulting chamber was then filled with 1 mL PBS and 100 μL of untreated, heat-inactivated, or bleach-treated oocyst suspension were seeded onto the PLL treated zone and let to settle for 45min to 1 hr at RT. Three gentle rinsing steps, with 1mL PBS, were performed before mounting on the AFM. We observed that this procedure did not grossly affect the different sub-populations ratios as compared to the original suspension (Fig. S5B).

Observation lasted for 2 hr maximum at RT with occasional supplementations of water to counteract the evaporation. (right) oocysts were imaged together with the AFM cantilever (i.e. the dark triangularshape object on the pictures), showing that one can distinguish them easily while doing AFM. The presence of other fecal objects such as yeasts or larger objects (mainly fiberlike debris, arrow) invariably occurred because the oocysts we used for AFM experiments were extracted and stored in water with no additional chemicals to limit bacterial proliferation. Such non-target objects could be located near the oocysts, at the same focal plane, however they did not affect the overall AFM cantilever motions, except on rare occasions. In these latter cases, the corresponding force curves were not processed for fur-

  . Corresponding immunofluorescence and AF representative images are shown in SI Appendix, Fig. S2. Between 21.2 and 25.6% of H 2 O-exposed oocysts were labeled with antibody mAb 4B6

  From a methodological point of view, this study proposes a simple but efficient way to immobilize hardened biological microparticles such as T. gondii oocysts on glass slides for investigating the biophysical properties of their multilayer wall by using AFM and 14 27 fluorescence microscopy techniques. It opens the possibility to extend such studies to immobilized T. gondii sporocysts and to other environmentally-resistant parasitic pathogens such as Cryptosporidium and Giardia[START_REF] Byrd | Interaction force profiles between Cryptosporidium parvum oocysts and silica surfaces[END_REF][START_REF] Byrd | Investigation of the interaction force between Cryptosporidium parvum oocysts and solid surfaces[END_REF](55)[START_REF] Considine | Laterally-resolved force microscopy of biological microspheres-oocysts of Cryptosporidium parvum[END_REF].

15 29

 15 Chemical and Physical Treatment of the OocystsBleach Treatment. H 2 O-stored oocysts were washed three times in PBS at 5000g 5 min and then incubated with 1 mL of bleach solution containing 3% sodium hypochlorite (Fouque Chimie Service, Marseille, France) in PBS for 30 min at 4°C. The oocysts were then washed three times in PBS to remove bleach prior to be immobilized on coverslips for AFM experiments.Heat Inactivation. H 2 O-stored or bleach-treated oocysts were washed three times in PBS at 5000 g for 5 min, resuspended in 500 µL PBS and then placed in a dry block heater for 10 min at 80°C to allow their inactivation prior to AFM experiments.

Figure 1 : 56 Figure 2 :

 1562 Figure 1: Ultrastructure of the T. gondii oocyst wall. (A) Low magnification image of a control (water maintained) oocyst showing the ruptured oocyst wall (OW) and the remnants of a sporocyst (Sp). Bar is 1 µm. (B) Detail of the oocyst wall from a control oocyst showing the thinner outer layer (O) and the thicker inner layer (I). Bar is 100 nm. (C) Detail of the oocyst wall from a bleached oocyst showing the inner layer (I) with loss of the outer layer except for a few remnants (arrows). Bar is 100 nm. (D) Detail of the sporocyst wall from a control oocyst showing the outer (O) and inner (I) layers of the wall. Bar is 100 nm. (E) Detail of the sporocyst wall from a bleached oocyst showing the retention of both the outer (O) and inner (I) layers for the wall. Bar is 100 nm.

Figure 3 :

 3 Figure 3: Measuring mechanical properties of T. gondii oocysts. (A) Typical force curve (Force vs. Tip-sample separation) used for quantifying the Young modulus (note that pressing and pulling curves are almost superimposed). The dotted line is the point of contact. (B) Zoom on the contact region of the curves presented in A, showing the superimposition of pressing and pulling curves together with the Hertz fit described in the text (dotted line), and d the maximal indentation. The superimposition of pressing and pulling curves shows that little if no dissipation is occurring in the material when indenting, ruling out any viscoelastic behavior under the conditions of our experiments. (C) Example of maximal indentation for untreated oocysts of different subtypes, showing no tendency upon repeated indentation. Note that 100 nm was used as the maximum of the scale as it is the upper bound of the thickness of the oocyst wall found in literature. (D) Maximal indentation under a force of 1 nN as a function of oocyst subtype and treatment. (E) Repeated measured values of Young modulus of 3 untreated oocysts of various subtypes,

Figure 4 :

 4 Figure 4: Measuring non-specific adhesive properties of T. gondii oocysts. (A) Typical force curve showing no adhesion event. (B) Typical force curve showing adhesion event. We present here the zoom on the force curve near the contact / adhesion zone, with the recorded maximal adhesion force. Note that the noise is far below the forces measured (typically 15 to 30 pN). (C) The fraction of adhesion as a function of oocyst subtypes and treatments (mean +/-SD over the different days of experiments). Arrows show cases where only one object for a given condition was observed over different repetitions. Dotted line has been placed at 75 % adhesion as a guide for the eye. (D) Same data as in C, showing the comparison of NS and SP oocysts as a function of treatment. (E) Example of maximal separation force for untreated oocysts of different subtypes, showing no tendency upon repeated indentation / pulling, indicating that no strong pollution of, or transfer of material, to the lever tip was observed. Note that the dotted line corresponds to the average observed noise on the baseline of the force curves. (F) Distribution of adhesion forces as whisker plots for oocysts of different subtypes and treatments. The significant differences correspond to the high / low adhesion separation on the graph (SI Appendix, TablesS3 and S4).

36 71Figure S2 :Figure S3 : 41 81Figure S4 : 84 Figure S5 :

 36S2S341S484S5 Figure S2: Fluorescence patterns of the wall of T. gondii oocysts exposed solely to H 2 O (A), heated at 80°C (B), treated by bleach solution (C), or bleach-and then heat-treated (D) as described in the materiel and methods section. Oocysts were then allowed to react with 4B6 antibody specific to their inner wall (4, 5). BF, bright field; AF, autofluorescence under UV excitation (red channel); FITC corresponds to 4B6 fluorescence (green channel). Merge presents overlay between AF and FITC channel. Scale bars = 5 µm.

Figure S6 :

 S6 Figure S6: Proposed structure of the bi-layered wall of the Toxoplasma oocyst in terms of mechanics and adhesive properties. Temperature and bleach treatments have no effect on the oocyst wall mechanics but have opposite effect on wall adhesion and accessibility to inner wall by a specific antibody: bleached oocysts exhibit higher adhesion frequency and forces than heated or control oocysts. Little differences are observed among the different maturing stages.

Table S2 : Statistical analyses of the Young modulus as a function of oocyst subtype and treatment

 S2 (see Fig.3F). Data obtained with Prism 6 (GraphPad).

Table S3 : Statistical analyses of the distribution of adhesion forces as whisker plots for oocysts of different subtypes and treatments (see Fig. 4F

 S3

). Data obtained with Prism 6 (GraphPad).

Table S4 : Summary of median values of indentation, E moduli, and adhesion force of Toxoplasma oocysts at different maturing stages following different surface treatments

 S4 . n = conserved data points.

	1245
	1246
			H2O			Heat			Bleach			Bleach→heat
		NS	SB	SP	NS	SB	SP	NS	SB	SP	NS	SB	SP
	Indentation	22,5	22,6	20,5	11,4	8,60	12,4	17,0	15,0	18,5	16,8	23,9	17,1
	(nm)
	n (oocysts)	9	5	18	11	1	27	5	1	8	6	4	22
	E modulus	5.3	2.8	4.2	11.1	22.2	11.1	7.9	14.0	5.7	8.8	5.0	6.0
	(MPa)
	n (oocysts)	9	5	19	13	1	33	5	1	8	6	5	24
	Adhesion force	85,0	79,5	75,5	86,0	100,0	94,5	184,5	134,5	246,0	244,5	197,0	234,0
	(pN)
	n (force	67	34	124	51	7	186	46	8	90	60	53	187
	curves)
	115												58

Acknowledgments

We gratefully thank M.L. Dardé (INSERM U 1094 NET, Limoges University, France) for providing 4B6 hybridoma, and P.A.R.I.S. Biotech (Compiègne, France) for antibody production and purification. This work was supported by Aix-Marseille University (Préciput 2011 program). A.D. and N.A. are currently supported by the ANR Protofood program, and P.-H.P. by the ANR JCJC DissecTion program.

Data Processing and Statistical Analysis

The distributions of the pooled data being non gaussian, median values with data points and/or whisker plots were then plotted as a function of the oocyst stage and treatment using Prism 5.0 and 6.0 (GraphPad). The data sets were compared using non-parametric tests such as Kruskal-Wallis.