Jonathan A C Roques
email: jonathan.roques@bioenv.gu.se

Camille Houdelet

Freddie-Jeanne Richard

Stress response in terrestrial isopods: a comparative study on glycaemia

Keywords: bioindicator, glyphosate, acute stress, social isolation, Armadillidium vulgare, Porcellio dilatatus

Stressors are omnipresent in the environment and trigger specific physiological and/or behavioural responses that remain relatively less known in invertebrates. Among the parameters used to quantify the impact of stress on physiology, the circulating level of glucose is commonly used in aquatic crustacean species. Terrestrial isopods are small crustaceans commonly used as bioindicators of soil quality; however, their response to environmental stressors remains poorly documented. In laboratory conditions, we investigated how different stressors influence the physiological response of two terrestrial isopod species. Thus, we exposed Armadillidium vulgare and Porcellio dilatatus to a series of both acute (physical) and chronic (temperature, social isolation, and exposure to herbicide) stressors that mimic potentially stressful situations encountered in their environment. Our results revealed that, for both species, glycaemia is not impacted by thermic shock (1 h at 4°C), mechanical disturbances (1 minute of shaking or upside-down motion) or social isolation (4 weeks) at the population level. Nevertheless, we observed a significantly higher sensitivity in male P. dilatatus exposed to upside-down movements and in males of both species exposed to social isolation. Acute (96 hours) and chronic (21 days) exposure to glyphosate (1.8-2.5 kg.ha -1 ) did not impact glycaemia levels in A. vulgare. However, at both tested doses, individuals exposed to glyphosate for 25 days and subjected to an additional stressor (haemolymph sampling at 96 hours) had significantly higher glycaemia in comparison to their control. Survival was also impacted by the joint application of chronic glyphosate exposure and intermediate haemolymph sampling. We conclude that glucose is a potentially good indicator to study the stress response of terrestrial isopods. Additional studies for this parameter, combined with other physiological indicators, are needed to better characterize this response.

Introduction

In ecology, the ability of an organism or a population to respond to a given change can be used as a biological indicator, also called a bioindicator, of the quality of an environment [START_REF] Whitfield | Fishes as indicators of environmental and ecological changes within estuaries: a review of progress and some suggestions for the future[END_REF]. For instance, bioindicator species can be used to measure and evaluate the impact of potentially harmful chemicals, such as heavy metals or pesticides, on the environment [START_REF] Bargańska | Honey bees and their products: bioindicators of environmental contamination[END_REF][START_REF] Hopkin | The isopod Porcellio scaber as a monitor of the bioavailability of metals in terrestrial ecosystems: towards a global 'woodlouse watch' scheme[END_REF]. Changes can be measured at different levels, such as specific diversity and abundance [START_REF] Santorufo | Soil invertebrates as bioindicators of urban soil quality[END_REF], or at the individual level with records of mortality and fecundity [START_REF] Jones | Reproductive allocation in the terrestrial isopods Porcellio scaber and Oniscus asellus in a metal-polluted environment[END_REF], physiology [START_REF] Folguera | An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming[END_REF] or behaviour (Zidar et al., 2005) to evaluate the direct impact of environmental changes. To date, several vertebrate and invertebrate species have been identified as reliable bioindicators of their natural habitat [START_REF] Bargańska | Honey bees and their products: bioindicators of environmental contamination[END_REF][START_REF] Carravieri | Penguins as bioindicators of mercury contamination in the Southern Ocean: birds from the Kerguelen Islands as a case study[END_REF][START_REF] Gerlach | Terrestrial invertebrates as bioindicators: an overview of available taxonomic groups[END_REF][START_REF] Paoletti | Woodlice (Isopoda: Oniscidea): their potential for assessing sustainability and use as bioindicators[END_REF][START_REF] Park | Mitigating the impacts of agriculture on biodiversity: bats and their potential role as bioindicators[END_REF][START_REF] Pearce | Small mammals as bioindicators of sustainable boreal forest management[END_REF][START_REF] Souty-Grosset | Investigations on the potential of woodlice as bioindicators of grassland habitat quality[END_REF]. Among invertebrate bioindicator species, terrestrial isopods have the potential to be used as indicators in both moist areas and grasslands [START_REF] Gerlach | Terrestrial invertebrates as bioindicators: an overview of available taxonomic groups[END_REF][START_REF] Souty-Grosset | Investigations on the potential of woodlice as bioindicators of grassland habitat quality[END_REF]. Terrestrial isopods are important decomposers of leaf litter, and their integrity may be rapidly threatened when a disturbance occurs in the soil [START_REF] Dallinger | Terrestrial isopods: useful biological indicators of urban metal pollution[END_REF][START_REF] Souty-Grosset | Investigations on the potential of woodlice as bioindicators of grassland habitat quality[END_REF].

When an organism is threatened, its resources are mobilized and redirected from functions such as growth or reproduction to face the challenges and preserve its integrity [START_REF] Moberg | The biology of animal stress: basic principles and implications for animal welfare[END_REF]. This response is known as the stress response. Invertebrates are frequently exposed to a number of stressors, including repeated predation attempts [START_REF] Hawlena | Grasshoppers alter jumping biomechanics to enhance escape performance under chronic risk of spider predation[END_REF], adverse environmental conditions [START_REF] Chown | Insect physiological ecology: mechanisms and patterns[END_REF] and parasitism [START_REF] Chang | Crustacean hyperglycemic hormone and hemolymph metabolites: Stress responses in two lobster species[END_REF]. As stressors are omnipresent in the environment, the stress response is essential for the survival of an individual and relatively well preserved during the course of evolution [START_REF] Nesse | Evolutionary origins and functions of the stress response system[END_REF]. Reactions towards stress are highly variable not only among different species but also within individuals of the same species [START_REF] Koehn | Towards a physiological and genetical understanding of the energetics of the stress response[END_REF]. As the stress response is initially adaptive and protective, prolonged exposure to a given stressor or a combination of stressors can further evolve into maladaptive responses and be detrimental for an organism or a community [START_REF] Moberg | The biology of animal stress: basic principles and implications for animal welfare[END_REF]. Stress can cause severe damage to an organism with a negative impact on its survival and fitness and potential consequences at the population level [START_REF] Hoffmann | Environmental stress as an evolutionary force[END_REF]. As an example, the accumulation of various stressors resulted in the recent worldwide increase in colony failure rates, or 'colony collapse disorder', in honeybee [START_REF] Even | General stress responses in the honey bee[END_REF].

The stress response has been extensively studied in vertebrates and is the result of physiological changes in response to stressful stimuli [START_REF] Kassahn | Animal performance and stress: responses and tolerance limits at different levels of biological organisation[END_REF]. The stress response can be measured at three levels. The primary stress response is the release of stress hormones, such as corticosteroids and catecholamines, in the bloodstream [START_REF] Tsalafouta | Ontogenesis of the HPI axis and molecular regulation of the cortisol stress response during early development in Dicentrarchus labrax[END_REF]. The main role of these compounds is to restore homeostasis following exposure to stressors. The secondary stress response acts at the cellular level by mobilizing and reallocating energy and increasing cardiac output and oxygen uptake and transfer. In mammals, for example, cortisol can facilitate the increase in the production and availability of metabolites such as glucose. Gluconeogenesis is promoted in the liver, while glucose use by the cells is limited to increase blood glucose availability to meet the needs of the organism in response to stressful stimuli [START_REF] Desborough | The stress response to trauma and surgery[END_REF]. The tertiary response affects the entire organism and can inhibit functions such as growth, reproduction, immune response and the ability to face additional stressors [START_REF] Moberg | The biology of animal stress: basic principles and implications for animal welfare[END_REF][START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions 1[END_REF][START_REF] Tsigos | Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress[END_REF].

Although invertebrates are susceptible to stressors, the stress response in invertebrates has been studied to a lesser extent than it has in vertebrates. Therefore, fewer cellular and physiological indicators of stress in invertebrates are known. At the cellular level, the increased production or activation of antioxidant proteins and heat shock proteins is seen in response to a stressor, which is similar to the vertebrate response [START_REF] Feder | Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology[END_REF].

Relatively few invertebrate species have been studied regarding the stress response. Among them, we find some model species for biological processes, such as Caenorhabditis elegans or Drosophila melanogaster [START_REF] Feder | Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology[END_REF], or economically and ecologically important species, such as the honeybee Apis mellifera [START_REF] Even | General stress responses in the honey bee[END_REF] and aquatic crustacean species, such as the American lobster Hommarus gammarus [START_REF] Chang | Crustacean hyperglycemic hormone and hemolymph metabolites: Stress responses in two lobster species[END_REF] or the white leg shrimp Litopenaeus vannamei [START_REF] Chang | Cold shock-induced norepinephrine triggers apoptosis of haemocytes via caspase-3 in the white shrimp, Litopenaeus vannamei[END_REF]. In aquatic crustacean species, it has been shown that exposure to harmful physical or chemical stimuli resulted in an elevation of glucose haemolymph [START_REF] Lorenzon | Hyperglycemic stress response in Crustacea[END_REF][START_REF] Lorenzon | Lipopolysaccharide-Induced Hyperglycemia Is Mediated by CHH Release in Crustaceans[END_REF]. These physiological changes were shown to be under the control of the crustacean hyperglycaemic hormone (CHH) [START_REF] Chang | Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses[END_REF][START_REF] Chang | Crustacean hyperglycemic hormone and hemolymph metabolites: Stress responses in two lobster species[END_REF][START_REF] Lorenzon | Hyperglycemic stress response in Crustacea[END_REF][START_REF] Lorenzon | Lipopolysaccharide-Induced Hyperglycemia Is Mediated by CHH Release in Crustaceans[END_REF]. This hormone is secreted from the sinus gland [START_REF] Fanjul-Moles | Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: review and update[END_REF] in response to stressful stimuli in crustaceans [START_REF] Chang | Crustacean hyperglycemic hormone and hemolymph metabolites: Stress responses in two lobster species[END_REF]. Moreover, this hormone has been described as the crustacean equivalent of cortisol and corticosterone [START_REF] Elwood | Pain and stress in crustaceans?[END_REF].

Terrestrial isopods have been used in tests of the toxicity of soil, and the parameters of evaluation were mainly the abundance of individuals [START_REF] Faulkner | Increased abundance of terrestrial isopod populations in terrestrial ecosystems contaminated with petrochemical wastes[END_REF], reproduction rate [START_REF] Niemeyer | Reproduction of Cubaris murina (Crustacea: Isopoda) under laboratory conditions and its use in ecotoxicity tests[END_REF] and survival [START_REF] Stanek | Linkage of biomarkers along levels of biological complexity in juvenile and adult diazinon fed terrestrial isopod (Porcellio scaber, Isopoda, Crustacea)[END_REF]. Porcellio scaber were studied most often regarding the metal bioaccumulation process [START_REF] Da Silva Souza | Morphological biomarkers in the Rhinocricus padbergi midgut exposed to contaminated soil[END_REF]. In this study, we chose to investigate the impact of several potential stressors on the glucose levels of circulating haemolymph in two terrestrial isopod species, Armadillidium vulgare (Latreille, 1804) and Porcellio dilatatus (Brandt, 1833). These species belong to two different ecomorphological groups, with different defence strategies: A. vulgare is a roller and P. dilatatus is a clinger [START_REF] Schmalfuss | Eco-morphological strategies in terrestrial isopods[END_REF]. In their habitat, these organisms can be exposed to various stressors that can be biotic or abiotic and acute or chronic. The main stressors encountered can be separated into three categories: 1) biological stressors (predators, parasites, fungi, bacteria and viruses); 2) chemical stressors (pesticides, fungicides, and herbicides), which can cause damage to non-target species; and 3) environmental stressors (agricultural practices, (micro)climate change, resource abundance fluctuation, social interactions, humidity, etc.). In our laboratory, we investigated the impact of five different stimuli that mimicked the whole range of potentially stressful situations encountered in the natural habitat or cultivated areas of these species [START_REF] Swan | Biodiversity and community composition in urban ecosystems: coupled human, spatial, and metacommunity processes[END_REF][START_REF] Szlavecz | Terrestrial isopods in urban environments: an overview[END_REF]. The main objective of the present study was to assess the importance of three extrinsic factors (temperature, shaking, and falling), one intrinsic factor (gender) and social privation on the glycaemia level.

The approach of using more subtle endpoints, such as physiological and behavioural changes, to assess the toxicity of potentially harmful substances has become more popular and has been shown to be more accurate than the traditional survival, growth and reproduction responses [START_REF] Drobne | Terrestrial isopods-a good choice for toxicity testing of pollutants in the terrestrial environment[END_REF]. In line with this approach, we use glycaemia as a physiological endpoint to measure the potential stress responses in two terrestrial isopod species.

Material and methods

Animals

The terrestrial isopods A. vulgare and P. dilatatus were derived from two natural populations collected in France (Nice, Alpes Maritimes, 1967, for A. vulgare andRom, Deux-Sèvres, 1988, for P. dilatatus). The animals were maintained in standard laboratory conditions (in boxes of moistened compost, at 20°C, and with the natural photoperiod of Poitiers, France 46°40'N) and fed ad libitum fresh organic carrots and dried leaves of linden (Tilia sp.). For Experiments 1 and 2, we selected one-year-old adult A. vulgare and P. dilatatus of similar size (around 60 mg and 150 mg respectively). For Experiment 3, we selected a mixture of virgin 1-to 3-year-old A. vulgare individuals (60-150 mg). All animals used were in intermoult (di-ecdysis or beginning the pre-ecdysis stages C/D0) (for growth cycle details, see [START_REF] Beauché | The best timing of mate search in Armadillidium vulgare (Isopoda, Oniscidea)[END_REF][START_REF] Beauché | The best timing of mate search in Armadillidium vulgare (Isopoda, Oniscidea)[END_REF]Zidar et al., 1998). The period of each experiment was chosen to match the moult cycle of each species (at least 26 days for both species; [START_REF] Beauché | The best timing of mate search in Armadillidium vulgare (Isopoda, Oniscidea)[END_REF]Zidar et al., 1998) in order to avoid moulting during the course of experiment. None of the tested individuals were moulting during the course of the experiment. Gravid females were excluded from the experimental pool. The experimental procedures and animal manipulations did not require an ethics statement. The stimuli used in these experiments were carefully selected to elicit a subtle response without causing any unnecessary distress.

Experimental procedure

All procedures were carried out in laboratory conditions during daylight hours between 10:00 and 16:00 in the spring and summer of 2016. We chose five different stimuli to be applied to the isopods: "cold", "shake", "upside-down" (Experiment 1), social isolation (Experiment 2) and chronic exposure to glyphosate (Experiment 3). The stimuli were chosen as they could mimic threats that the animals encounter in natural situations.

Experiment 1: biological stressor

Animals were randomly subjected to one of the three following acute stimuli: exposure to cold temperature, shaking or an upside-down movement. The cold stimulus was chosen as ectothermic animals, including terrestrial isopods, are sensitive to severe temperature changes as their body temperature is mostly dependent on the temperature of their environment [START_REF] Dahlhoff | The role of stress proteins in responses of a montane willow leaf beetle to environmental temperature variation[END_REF]. The shaking stimulus was used to mimic the impact of modern agricultural practices, such loud noises and vibration disturbances caused by heavy machinery [START_REF] Schlacher | Exposure of Fauna to Off-Road Vehicle (ORV) Traffic on Sandy Beaches[END_REF][START_REF] Soane | Soil compaction problems in world agriculture[END_REF]. The upsidedown stimulus aimed to mimic the impact of predator attacks. In nature, terrestrial isopods can be targeted by numerous predators which could catch, move and drop them [START_REF] Tuf | Personality affects defensive behaviour of Porcellio scaber (Isopoda, Oniscidea)[END_REF].

For each species, 20 undisturbed individuals (10 males and 10 females) were randomly picked from the holding tanks and served as control groups. The 'cold' stimulus consisted of placing two individuals in one box (Ø 8 cm, H 5 cm) kept at 4°C for one hour. The light condition was kept constant during this period (refrigerator with a glass door), and the box was kept moist using water-dipped tissues in the bottom). The "shake" stimulus consisted of shaking the animals individually in a box (Ø 8 cm, H 5 cm) attached to an IKA KS 130 Basic 2D shaker (IKA, Staufen, Germany) for one minute at 560 rpm. The "upside-down" stimulus consisted of placing the animals in individual boxes (Ø 9.5 cm, H 5 cm) and overturning the box repeatedly by performing ample movements (180°) from right to left for one minute.

This stimulus was carried out by the same experimenter at a constant speed (approximately 70 movements). Stimuli were applied randomly to different individuals.

Experiment 2: social isolation

We investigated the impact of social isolation, which could be considered a chronic stressor for the studied species. Terrestrial isopods are known to be gregarious organisms. Living in groups has been demonstrated to offer several advantages, such as the prevention of desiccation, the potential protection from predators and better access to mates and secondary food resources [START_REF] Broly | Benefits of aggregation in woodlice: a factor in the terrestrialization process?[END_REF]. For this stimulus, 36 animals per species (18 males and 18 females) were placed in individual boxes (Ø 8 cm, H 5 cm) and kept isolated for a period of 4 weeks. The boxes were visually isolated from each other by cardboard sheets. In parallel, 120 individuals 120 were sexed and kept in groups of 6 (20 boxes, Ø 11 cm, H 5 cm; 10 boxes for males and 10 boxes for females) and served as the controls. On the sampling day, the analyses of both the isolated and grouped individuals were performed simultaneously. In total, 20 animals (10 males, 10 females, one individual per box) were sampled.

Experiment 3: chemical stressor

The animals were chronically exposed to the glyphosate-based herbicide Roundup® (Monsanto, USA). Glyphosate-based herbicides are among the most widely used herbicides for weed control and have become one of the most commonly used agrochemicals worldwide [START_REF] Shang | Screening and Isolation of Bacteria Degrading Herbicide Glyphosate and Its Degradation Efficiency[END_REF]. Despite studies of the impact of glyphosate on several non-target species [START_REF] Balbuena | Effects of sublethal doses of glyphosate on honeybee navigation[END_REF][START_REF] Giesy | Ecotoxicological risk assessment for Roundup® herbicide, Reviews of environmental contamination and toxicology[END_REF], the sublethal effect remains poorly studied.

Terrestrial isopods have been proposed as good candidates to study the impact of pollutants in terrestrial environments [START_REF] Drobne | Terrestrial isopods-a good choice for toxicity testing of pollutants in the terrestrial environment[END_REF]. Animals (A. vulgare) were randomly exposed to one of three doses of glyphosate (0, 1.8 and 2.5 kg.ha -1 ) for up to 25 days. These doses are related to the mg of glyphosate present in the glyphosate-formulated herbicide Roundup®. In this study, we did not aim to observe high mortality but rather to test the impact of the doses recommended by the manufacturer and approved by the French Ministry of Agriculture and Food at the time of the experiment for use against easy-to-control (1.8 kg.ha -1 ) and difficult-to-control (2.5 kg.ha -1 ) weeds. Animals were subsequently placed in plastic boxes (Ø 10 cm, 5 cm H) containing 70 (±0.5) g of moistened compost. Each box contained 5 animals of the same sex and same age class (N=40/treatment). The animals were acclimated in the boxes for four days, during which they received their usual food. After this acclimatization period, the uneaten food was removed.

The glyphosate used in our study was in the form of isopropylamine salt (170 g.l -1 ) as specified in the Roundup® formulation. Animals were exposed to the desired dose of glyphosate (Roundup®) through the food and the soil. For each box, 5-6 pieces of thinly sliced carrot and the equivalent of two linden leaves were soaked with a mixture of 7 ml of water and 0 µl, 8.3 µl or 11.5 µl of Roundup® for 15 minutes prior to being placed in the appropriate box. The remaining solution was subsequently pipetted homogeneously into the box, where it was placed on the soil, not directly on the animals. This process was repeated weekly until the completion of the experiment (25 days maximum). Mortality was recorded during these processes, and dead individuals were removed from the experimental boxes. One group was sampled after 96 hours (acute exposure), and one group was sampled after 21 days (chronic exposure). In addition, after the puncture, the animals from the group sampled after 96 hours were kept moist in recovery boxes with water-dipped tissues at the bottom for 24 hours. Surviving individuals were placed back into their original experimental boxes and sampled after 21 additional days (25 days in total). This was done to investigate the effect of an extra stressor (haemolymph sampling, mimicking a wound) in addition to the chronic exposure to glyphosate.

Immediately after each stimulus, circulating haemolymph was sampled by making a puncture in the intersegmental membrane between the thorax and the abdomen. The respective control groups from each experiment were also sampled for haemolymph at the same time. In total, 5-10 μl of haemolymph per individual was collected using a micropipette and stored at -20°C until further analyses. After the puncture, the animals were kept moist in recovery boxes using water-dipped tissues on the bottom for 24 hours. Surviving individuals (>50%) were placed back into their original housing boxes with moistened compost litter and were not further used for another study.

Glucose determination

Haemolymph glucose was measured with a commercially available enzymatic test kit (Glucose HK, Sigma-Aldrich, St. Louis, USA), with protocols adapted to a 96-well microplate [START_REF] Schram | The impact of elevated water ammonia concentration on physiology, growth and feed intake of African catfish (Clarias gariepinus)[END_REF]. Samples are first diluted in distilled water (1:6); then, 10 μl of the diluted sample or standard (5.55 mmol.l -1 glucose) are mixed with 200 μl of the reagent provided with the kit and incubated for 15 minutes at 20°C. Absorbance was read within 30 minutes at 340 nm (Berthold LB941 Multimode reader, Berthold Technologies, Zug, Switzerland).

Data analyses and statistics

Data are summarized by box and whisker plots that present first, second (median), and third quartiles as horizontal lines in the box and upper and lower values as "whiskers" extending from the boxes; In some instances, not all samples collected were analysed because of insufficient haemolymph quantity. For Experiment 1 (the "cold" and "shake" stimuli) and Experiment 3, there were no gender differences, and, therefore, the data were pooled.

However, for the "upside-down" stimulus (Experiment 1) and Experiment 2, the data were split between males and females. The data were not normally distributed; therefore, the nonparametric Mann-Whitney test was used to assess statistical significance for all the experiments. We used the Chi-square test to compare the survival of individuals in Experiment 3.

Results

Experiment 1: biological stressor

There were no differences in the glucose haemolymph concentration regardless of the species or the type of stimulus tested in comparison with the control condition when the data were pooled (Fig 1A and1B) (Mann-Whitney test; for P. dilatatus and A. vulgare, respectively: "cold": W=59, p=0.32; W=151, p=0.27; "shake": W=93.5, p=0.41; W=141, p=0.17; "upsidedown": W=100, p=0.16; W=180.5, p=0.79). For A. vulgare, no gender-specific differences were found (Mann-Whitney test, p> 0.05 for all stimuli, Fig 1B and2B). Similarly, P. dilatatus subjected to the cold and shake stimuli did not display gender differences regarding their glucose haemolymph (Fig 1A). However, male P. dilatatus had significantly haemolymph glucose levels compared to those of their female counterparts for the upsidedown stimulus only (Mann-Whitney test, W=20, p=0.03, Fig 2A). These levels were significantly higher than those of the male control group (Mann-Whitney test, W=13, p=0.02,

Fig 2A).

Experiment 2: social isolation

There were no differences in the glucose haemolymph concentration for either species in comparison with their respective controls when the data were pooled (Mann-Whitney test; W=106.5, p=0.20 for P. dilatatus and W=157, p=0.08 for A. vulgare). However, we observed a gender difference for both species: male P. dilatatus and A. vulgare subjected to social isolation for a month had significantly higher haemolymph glucose levels compared to those of their respective control groups (Mann-Whitney test, W=56, p=0.04 for P. dilatatus,; and W=14.5, p<0.01 for A. vulgare, Fig 3A and3B). No differences in glucose haemolymph were found between females subjected to social isolations and females kept in group for either species (Mann-Whitney test, W=98, p=0.96 for P. dilatatus and W=53, p=0.47 for A. vulgare).

Experiment 3: chemical stressor

There was no effect on the glucose haemolymph concentration when A. vulgare individuals were exposed to either dose of glyphosate for 96 hours (Mann-Whitney test, W=506.5, p=0.47 for the 1.8 kg.ha -1 group and W=475, p=0.21 for the 2.5 kg.ha -1 group, Fig 4A ) or 21 days (Mann-Whitney test, W=353,p=0.45 and W=295.5,p=0.18,Fig 4B). However, individuals who were sampled after 25 days of exposure to either concentration and received another stressor (i.e., a haemolymph puncture after 96 hours) had significantly higher glucose haemolymph than that of the control group (Mann-Whitney test, W=57, p=0.01 for the 1.8 kg.ha -1 group and W=64.5, p=0.05 for the 2.5 kg.ha -1 group, Fig 4C). Survival tended to decrease naturally with time in all groups (non-significant; X 2 =0.13, ddl=2; X 2 =0.33, ddl=2; and X 2 =0.58, ddl=2 for the control group and the 1.8 and 2.5 kg.ha -1 glyphosate groups, respectively) (Fig 5). Survival was only negatively affected by the combination of chronic exposure to glyphosate and the application of an extra stressor (Fig 5). No differences in survival were observed between individuals from the control group samples after 96 hours and those from the group exposed to glyphosate for 96 hours + 21 days (X 2 =3.63, ddl=1), while survival decreased in both the 1.8 and 2.5 kg.ha -1 glyphosate groups (X 2 =6.23, ddl=1 and X 2 =7.41, ddl=1, respectively). There were no differences in survival among all groups after 96 hours and 21 days of exposure (X 2 =0.58, ddl=1; X 2 =0,01, ddl=1; and X 2 =0.053, ddl=1 for the control group and the 1.8 and 2.5 kg.ha -1 glyphosate groups, respectively) (Fig 5).

Discussion

The elevation of circulating glucose levels is a common response to stressors and is found throughout the animal kingdom, including crustaceans. Crustaceans have been widely used for stress risk assessment and are used as bioindicators or biomonitors, especially in various aquatic systems [START_REF] Rinderhagen | Crustaceans as bioindicators, Biomonitoring of Polluted Water-Reviews on Actual Topics[END_REF]. Among commercial aquatic crustaceans species, stress exposure, such as emersion, transport and handling, is common and causes hypoxia and variation in physiology [START_REF] Lorenzon | Hyperglycemic stress response in Crustacea[END_REF]. For example, the decapods, Liocarcinus depurator and Munida rugosa showed a higher concentration of glucose when subjected to trawling in comparison to creel-caught crustaceans [START_REF] Bergmann | Physiological stress in decapod crustaceans (Munida rugosa and Liocarcinus depurator) discarded in the Clyde Nephrops fishery[END_REF]. Another study revealed that the CHH hormone concentration increased in the haemolymph in less than 1 hour after acute stress (handling) in lobsters, which is slower than the appearance of biogenic amines [START_REF] Chang | Crustacean hyperglycemic hormone and hemolymph metabolites: Stress responses in two lobster species[END_REF]. The production and action of these hormones leads to mobilization of energy reserves, such as glucose. In the current study, we used two terrestrial isopod species to test the glucose level variation in response to different stressors.

We found that stress induced in A. vulgare and P. dilatatus adults by the cold and shake stimuli had no significant impact on the haemolymph glucose levels, which were collected right after the application of the stimuli to test the short-term impact of our stressors.

When exposed to the upside-down acute stressor, we observed that males of P. dilatatus had significantly higher glucose levels than those of their controls, while there was no effect on male A. vulgare. These two species have different defence strategies. A. vulgare can roll and may isolate themselves from the exterior [START_REF] Schmalfuss | Eco-morphological strategies in terrestrial isopods[END_REF]. However, P. dilatatus corresponds to the clinger ecomorphological group [START_REF] Schmalfuss | Eco-morphological strategies in terrestrial isopods[END_REF] and is highly responsive to stimuli, but the duration of response (tonic immobility) is relatively short, with no difference between sex [START_REF] Quadros | Tonic immobility in terrestrial isopods: intraspecific and interspecific variability[END_REF].

In the second experiment, we observed a sex difference for both species when individuals were isolated for one month. The glucose levels of male are apparently affected, while those of females are not. These results indicate that male isopods, regardless of their defence strategies, are more sensitive to non-physical and chronic stress, such as social isolation, than their female counterpart. Here, we showed that different types of stress have different impacts between species, but we also observed differences in sensitivity between females and males.

Differences in sensitivity between sexes exist and have been previously observed in several invertebrates and in both aquatic and terrestrial systems [START_REF] Sornom | Effect of gender on physiological and behavioural responses of Gammarus roeseli (Crustacea Amphipoda) to salinity and temperature[END_REF] and reference therein [START_REF] Mcclellan-Green | Does gender really matter in contaminant exposure? A case study using invertebrate models[END_REF]Wilczek et al., 2008;Zeng et al., 2011). For example, under oxidative stress conditions, male flies ingested and excreted less food than females (Zeng et al., 2011). In two species of spider, cellular reactions against stress caused by metal pollutants seemed to be sex-dependant (Wilczek et al., 2008). In the terrestrial isopod P. scaber, a close relative of P. dilatatus, an exposure to dimethoate for 48 hours impacted the locomotor behaviour (increased hyperactivity and suppressed turning rate) of males only [START_REF] Bayley | Prolonged effects of the insecticide dimethoate on locomotor behaviour in the woodlouse, Porcellio scaber Latr.(Isopoda)[END_REF].

In the third experiment, we evaluated the effect of two recommended concentrations of glyphosate used in agriculture (1.8 and 2.5 kg.ha -1 ). Glyphosate ingestion might affect the learning and retrieval of memory for the recognition of food [START_REF] Balbuena | Effects of sublethal doses of glyphosate on honeybee navigation[END_REF], and chronic exposure to traces of glyphosate reduces the responsiveness to sucrose in honeybee [START_REF] Herbert | Effects of field-realistic doses of glyphosate on honeybee appetitive behaviour[END_REF]. Glyphosate is also known to reduce growth in the earthworm [START_REF] Springett | Effect of repeated low doses of biocides on the earthworm Aporrectodea caliginosa in laboratory culture[END_REF] and affect reproduction and development in the freshwater snail [START_REF] Tate | Effect of glyphosate on the development of Pseudosuccinea columella snails[END_REF]. In our experiment, we observed no significant impact on A. vulgare glycaemia.

However, individuals who were chronically exposed to glyphosate and an additional stressor (intermediate haemolymph sampling) had altered glucose levels in comparison with individuals that only received the additional stressor without glyphosate. No individual moulted between the two sampling sessions. Stressors are omnipresent in the environment and can potentially interact with each other and affect the capacity of an organism to cope with a specific stressor [START_REF] Sornom | Effect of gender on physiological and behavioural responses of Gammarus roeseli (Crustacea Amphipoda) to salinity and temperature[END_REF]. While assessing the sublethal impact of a contaminant on an organism, it is important to take into account that the potential of an organism to cope with stressors and survive is determined by the ability of all its biological systems to work in concert [START_REF] Hebel | Responses of crustaceans to contaminant exposure: a holistic approach[END_REF]. For instance, here, we observed no direct impact of exposure to the recommended doses of glyphosate, but when animals were exposed to an additional stressor (haemolymph puncture) that can be compared to a minor wound, we observed that its combination with glyphosate exposure resulted in stress.

In conclusion, changes in glycemia can be used to assess stress in terrestrial isopods. Males of both species seemed to be more sensitive than female to stressors such as upside-down movements or social isolation. In our study, we have focussed on the very short term impact of single stressor. The effect combined stressors and the impact of glyphosate exposure on the middle and longer term require further investigations.

Figure legend Fig 1 .

 legend1 Figure legend

Fig 2 .

 2 Fig 2. Haemolymph glucose concentrations (whisker plot) in Porcellio dilatatus (A) and Armadillidium vulgare (B) in the control situation and one minute of repeated manual overturns in a box ("upside-down"). NS: not significant, *: p<0.05 (Mann-Whitney test).

Fig 3 .

 3 Fig 3. Haemolymph glucose concentrations (whisker plot) in Porcellio dilatatus (A) andArmadillidium vulgare (B) kept in groups of 6 individuals ("control") or kept isolated ("social isolation") for a period of 4 weeks. NS: not significant, *: p<0.05, **: p<0.01 (Mann-Whitney test).

Fig 4 .

 4 Fig 4. Haemolymph glucose concentrations (whisker plot) in Armadillidium vulgare exposed to 0 (control), 1.8 and 2.5 kg.ha -1 of glyphosate for 96 hours (A), 21 days (B) and 25 days with an extra stressor (haemolymph sampling) after 96 hours (C) NS: not significant, *: p<0.05, Mann-Whitney test).

Fig 5 .

 5 Fig 5. Survival (%) of Armadillidium vulgare exposed to 0 (control), 1.8 and 2.5 kg.ha -1 of glyphosate for 96 hours (T=96 H), 21 days (T=21 D) and 25 days with an extra stressor (T=96 H + 21 D) after 96 hours. * : p<0.05 Chi-square test.

Acknowledgements

We are grateful to Alexandra Laffite who is in charge of terrestrial isopods management and welfare in the Laboratory. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. polluted with heavy metals. Ecotoxicol. Environ. Saf. 70, 127-137. Zeng, C., Du, Y., Alberico, T., Seeberger, J., Sun, X., Zou, S., 2011. Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster. Fly 5, 174-180. Zidar, P., Božič, J., Štrus, J., 2005. Behavioral Response in the Terrestrial Isopod Porcellio scaber (Crustacea) Offered a Choice of Uncontaminated and Cadmium-Contaminated Food. Ecotoxicology 14, 493-502. Zidar, P., Drobne, D., Štrus, J., 1998. Determination of moult stages of Porcellio scaber (Isopoda) for routine use. Crustaceana 71, 646-654.


Karim Hayoun

Jean-Charles Gaillard

Olivier Pible

Béatrice Alpha-Bazin #

Jean Armengaud
email: jean.armengaud@cea.fr

High-throughput proteotyping of bacterial isolates by double barrel chromatography-tandem mass spectrometry based on microplate paramagnetic beads and phylopeptidomics

Keywords: High-throughput proteotyping Word count: 4, 860 Character count: 27, 415 proteotyping, tandem mass spectrometry, isolates, microorganisms, taxonomy, sample preparation

Tandem mass spectrometry-based proteotyping of microorganisms presents several advantages over whole-cell MALDI-TOF mass spectrometry: because a larger number of signals are recorded with better accuracy and precision, the approach allows for the identification of microorganisms at more resolved taxonomic levels, and can easily manage complex samples. Additionally, the use of SP3 paramagnetic beads for cell lysis and protein cleanup simplifies sample preparation for proteotyping. Based on these features, we have developed and tested a 96-well plate platform for high-throughput proteotyping of a large variety of bacteria. We evaluated the performance of the platform in terms of bacterial load and found no cross-contamination between wells. Likewise, phylopeptidomics analysis revealed no alteration in the relative quantifications of microorganisms. Finally, we applied this new format for rapid proteotyping of a large set of dental isolates using double-barrel chromatography coupled to tandem mass spectrometry, which maximizes the number of spectra per unit of time. The procedure allowed us to establish whether these isolates were pure strains or mixtures of strains and to identify the microorganisms at the most resolved taxonomic level.

Significance

The rapid and accurate identification of microorganisms is a clinical priority in medical diagnostics; however, specimens containing mixtures of microorganisms are difficult to analyze and the procedure is time-consuming. Tandem mass spectrometry proteotyping allows the fast identification of complex mixtures of microorganisms, known or unknown, and can also establish the biomass ratio of each component. We describe here a new workflow for preparing microbial samples in a 96-well-plate format for tandem mass spectrometry proteotyping and document its advantages and limitations. We demonstrate that this new format coupled to a highly efficient double-barrel LC-MS/MS system allows proteotyping of 96 isolates in 55h.

Highlights

We developed a 96-well-based SP3 platform for high-throughput tandem mass spectrometry proteotyping.

Quantitative phylopeptidomics reveals that the 96-well platform yields an identical relative abundance of microorganisms to conventional in-tube methods.

The 96-well platform can accept a broad range of sample material: from 2.8 × 10 5 to 1.7 × 10 9 cells. Double-barrel LC-MS/MS system allows proteotyping 96 samples in only 55 h.

Introduction

Proteotyping is a typing method for taxonomic identification of organisms according to their specific protein profile acquired by mass spectrometry [START_REF] Grenga | Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns[END_REF]. Proteotyping can be performed by whole cell matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, an established technique for the identification of microorganisms based on the molecular mass profile of their low molecular weight and basic proteins. The technique is designed to query the mass spectrum acquired directly on the sample against a database containing profiles of thousands of previously recorded microorganisms [START_REF] Lavigne | Mass spectrometry: a revolution in clinical microbiology?[END_REF]. The approach enables the rapid and confident identification of, for example, bacterial pathogens for clinical diagnostics at low cost [START_REF] Karger | Current developments to use linear MALDI-TOF spectra for the identification and typing of bacteria and the characterization of other cells/organisms related to infectious diseases[END_REF], and can be used for discriminating subspecies as illustrated for

Francisella tularensis [START_REF] Durighello | Proteogenomic biomarkers for identification of Francisella species and subspecies by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry[END_REF] or Campylobacter coli [START_REF] Emele | Proteotyping as alternate typing method to differentiate Campylobacter coli clades[END_REF], and for identifying antibiotic resistance in specific cases [START_REF] Carvalhaes | Rapid detection of ceftazidime/avibactam resistance by MALDI-TOF MS[END_REF]. Yeast and fungi can be also identified with the same principle [START_REF] Patel | A Moldy Application of MALDI: MALDI-ToF Mass Spectrometry for Fungal Identification[END_REF]. The MALDI-TOF approach is not, however, universal, as the spectra of most environmental microorganisms have not yet been recorded and, more importantly, only pure microorganisms can be identified [START_REF] Lavigne | Mass spectrometry: a revolution in clinical microbiology?[END_REF] due to the low number of mass features recorded. One of the ways to address these challenges is to extend microorganism proteotyping by measuring a larger number of signals by tandem mass spectrometry [START_REF] Grenga | Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns[END_REF][START_REF] Karlsson | Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus[END_REF]. Proteotyping by tandem mass spectrometry relies primarily on shotgun proteomics, wherein proteins present in the sample are proteolyzed into small peptides with trypsin; the generated peptides are then resolved by reverse phase chromatography and their sequence established by tandem mass spectrometry.

The taxonomic information associated with the peptide sequences is subsequently analyzed to identify the taxa present in the sample based on taxon-specific peptide sequences. This approach has been exemplified with several bacteria [START_REF] Boulund | Typing and Characterization of Bacteria Using Bottom-up Tandem Mass Spectrometry Proteomics[END_REF], and is sufficiently powerful to differentiate phylogenetically closely related bacteria [START_REF] Karlsson | Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics[END_REF] and even cellular forms of the same bacterium (e.g., vegetative cells and spores) [START_REF] Mappa | Assessing the ratio of Bacillus spores and vegetative cells by shotgun proteomics[END_REF]. Another advantage of tandem mass spectrometry-based proteotyping is its applicability to mixtures of microorganisms in complex samples, such as blood cultures [START_REF] Berendsen | Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks[END_REF] or feces [START_REF] Abbondio | Fecal Metaproteomic Analysis Reveals Unique Changes of the Gut Microbiome Functions After Consumption of Sourdough Carasau Bread[END_REF], and in the latter example no cultivation step is required. It can also be used to assess the biomass contributions of microorganisms in a complex sample by relying on the global inter-taxa fractions of shared peptides, as described in the "phylopeptidomics" principle [START_REF] Pible | Estimating relative biomasses of organisms in microbiota using "phylopeptidomics[END_REF], and has utility for monitoring antibiotic resistance biomarkers [START_REF] Chen | Detection of Antimicrobial Resistance Using Proteomics and the Comprehensive Antibiotic Resistance Database: a Case Study[END_REF]. Using the shotgun approach, tandem mass spectrometry proteotyping can be applied without any a priori information, and is thus more appealing for identification than targeted mass spectrometry. For all of these reasons, tandem mass depth characterization of environmental microbial communities [START_REF] Armengaud | Next-generation proteomics faces new challenges in environmental biotechnology[END_REF].

The efficiency of tandem mass spectrometry proteotyping depends on the amount of generated peptides and, accordingly, sample preparation including protein extraction, purification and proteolysis, is a critical step in the process. In-gel [START_REF] Hartmann | Taking the shortcut for high-throughput shotgun proteomic analysis of bacteria[END_REF], in-solution [START_REF] Ludwig | Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies[END_REF] and filter-aided sample preparation [START_REF] Wisniewski | Filter Aided Sample Preparation -A tutorial[END_REF][START_REF] Zougman | Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis[END_REF] have been the most common approaches used in shotgun proteomics over the last decade. Hughes and co-workers [START_REF] Hughes | Ultrasensitive proteome analysis using paramagnetic bead technology[END_REF][START_REF] Hughes | Singlepot, solid-phase-enhanced sample preparation for proteomics experiments[END_REF] have recently proposed an alternative sample preparation approach based on paramagnetic beads, termed single-pot solid-phase-enhanced sample preparation (SP3), wherein proteins are fixed onto magnetic beads and the protein/bead complexes can be easily retained using a magnet for washing and cleanup. Once bound to the bead surface, proteins are digested directly, and the resulting peptides are released ready for LC-MS/MS analysis. We previously showed that SP3 improved proteotyping performance over in-gel and S-Trap sample preparation with regards to peptide and protein identification [START_REF] Hayoun | Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry[END_REF]. Indeed, in the SP3 protocol, reduction and alkylation of proteins may be omitted and a short 15-minute proteolysis is sufficient, thus shortening sample preparation for tandem mass spectrometry proteotyping. Sample preparation performed in tubes is time-consuming and labor-intensive when numerous samples have to be analyzed, and is thus not suited to high-throughput clinical diagnostics. Accordingly, the adaption of sample preparation from tube format to multi-well plate format is an attractive concept, and has been described for classical proteomic sample preparation requiring labeling [START_REF] Dayon | Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma[END_REF], enrichment [START_REF] Humphrey | High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform[END_REF], or purification using FASP [START_REF] Potriquet | A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry[END_REF][START_REF] Yu | Quick 96FASP for high throughput quantitative proteome analysis[END_REF], S-Trap [START_REF] Hailemariam | S-Trap, an Ultrafast Sample-Preparation Approach for Shotgun Proteomics[END_REF], and more recently SP3 [START_REF] Dagley | Universal Solid-Phase Protein Preparation (USP(3)) for Bottom-up and Top-down Proteomics[END_REF][START_REF] Gonzalez-Lozano | A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis[END_REF][START_REF] Müller | Automated sample preparation with SP3 for low-input clinical proteomics[END_REF]. Here, we explored the possibility of performing 96-well plate sample preparation for tandem mass spectrometry proteotyping of microbial samples and compared the proteotyping outcomes against a single-tube format. We assessed magnet performance, established the range of bacterial cell load, and tested for cross-contamination and the relative quantities of mixed organisms. Finally, we applied this new format coupled to a highly efficient double-barrel LC-MS/MS system for proteotyping a large set of dental isolates.

Materials and methods

Microbial culture, mixture preparation and sampling

Bacteria were cultured overnight under aerobic conditions at 30°C and 140 rpm agitation. Saccharomyces cerevisiae was obtained directly by dissolving 82 mg of baker's yeast (Lesaffre, France) in 25 mL of PBS (Gibco). The Mix3* asymmetric mixture of microorganisms (5.8 × 10 8 cells of B. subtilis, 3.5 × 10 7 cells of E. coli and 1.0 × 10 6 cells of S. cerevisiae) was prepared from a culture of B. subtilis (1.1 × 10 9 cells.mL -1 ), E. coli (1.0 × 10 9 cells.mL -1 ), and S. cerevisiae (3.0 × 10 7 cells.mL -1 ) at an OD600nm equal to 1.0. After mixing, cells were collected by centrifugation at 8,000 × g for 5 min and stored at -20°C until use. Dental microorganisms were collected from three healthy volunteers using a toothpick.

Bacillus subtilis ATCC6633 (American

The material from the three toothpicks was solubilized in 1.2 mL of PBS and, after thorough mixing, 10 µL of this solution was diluted into 1 mL of PBS. A volume of 100 µL was sprayed on lysogeny broth (1× and 0.1×), brain heart infusion broth (1× and 0.1×) and Columbia medium supplemented with 5% sheep blood (both from bioMérieux) agar plates and incubated overnight at 30°C.

Cell lysis

Protein extraction was performed according to [START_REF] Hayoun | Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry[END_REF]. Briefly, 60 µL of 1× lithium dodecyl sulfate solution (Thermo Scientific) supplemented with 5% βmercaptoethanol (v/v) was added per 1 mg of cell pellet. The sample was then incubated for 5 min at 99°C in a thermomixer (Eppendorf) and sonicated for 5 min in an ultrasonic water bath (VWR ultrasonic cleaner USC 300 T) working at 45 kHz and 200 W. Microorganisms were disrupted by bead beating in 2-mL screw-cap microtubes (Sarstedt) containing an equal amount (66.7 mg) of 0.1 mm silica beads, 0.1 mm glass beads and 0.5 mm glass beads as previously described [START_REF] Hayoun | Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry[END_REF]. Disruption was performed using a Precellys Evolution instrument (Bertin Technologies) operated at 10,000 rpm for 10 cycles of 30 s, with 30 s of pause between each cycle. Beads were removed by centrifugation at 16,000 × g for 1 min and the supernatant was transferred to a new microcentrifuge tube and then incubated at 99°C for 5 min.

SP3 proteolysis

A total of 500 µg of hydrophilic (ref. n°24152105050250) and hydrophobic (ref.

n°44152105050250) SP3 commercial solutions (Sigma-Aldrich) at 10 mg/mL were mixed and suspended in 100 µL of Milli-Q water. Beads were retained using a nickel-plated neodymium magnet (N42 grade, Supermagnete, Webcraft, GmbH; reference Q-40-20-10-N), rinsed twice with 200 µL of Milli-Q water and then resuspended in 100 µL of Milli-Q water to give a 10 µg/µL stock solution, which was stored at 4°C until use as recommended [START_REF] Hughes | Ultrasensitive proteome analysis using paramagnetic bead technology[END_REF].

For SP3 proteolysis, 40 µg of beads (4 µL) were added to 20 µL of cell lysate, followed by acidification with 12 µL formic acid and activation of beads with 204 µL CH3CN (85% final concentration). Proteins were then trapped using either MagnaBind (Thermo Scientific) or Smart2 MBS (Tecan) neodymium magnetic racks, or hand-held N42 magnet (Supermagnete).

After removal of supernatant, proteins were washed twice with 200 µL of 70% ethanol and once with 180 µL CH3CN. These steps for purifying proteins were performed at room temperature. A volume of 10 µL of digestion buffer comprising 1 µg/µL of Trypsin Gold (Promega) in 50 mM NH4HCO3 and supplemented with 0.01% of ProteaseMAX surfactant (Promega) was added to the beads. Purified proteins were digested for 15 min at 50°C, beads were then trapped, and the recovered digests were acidified with 1 µL of trifluoroacetic acid (TFA, 0.5% final concentration).

Liquid chromatography and tandem mass spectrometry

Peptides were identified either with a Q-Exactive HF (Thermo Scientific) or an LTQ-Orbitrap XL (Thermo Scientific) tandem mass spectrometer coupled to an ultimate 3000 nano LC system (Thermo Scientific), operated as described [START_REF] Klein | RNA-binding proteins are a major target of silica nanoparticles in cell extracts[END_REF][START_REF] Trapp | Digging Deeper Into the Pyriproxyfen-Response of the Amphipod Gammarus fossarum With a Next-Generation Ultra-High-Field Orbitrap Analyser: New Perspectives for Environmental Toxicoproteomics[END_REF]. Peptides (30 or 300 ng) were desalted on a reverse-phase PepMap 100 C18 µ-precolumn (5 mm, 100 Å, 300 mm i.d. × 5 mm, Thermo Scientific) before peptide separation on a nanoscale PepMap 100 C18 nanoLC column (3 mm, 100 Å, 75 mm i.d. × 50 cm, Thermo Scientific) at a flow rate of 0.3 µL.min -1 using a 60 min gradient (2.5% B from 0 to 3 min, 2.5-25% B from 3 to 53 min and 25-40% B from 53 to 63 min) of mobile phase A (0.1% HCOOH/100% H2O) and phase B (0.1% HCOOH/80% CH3CN). The Q-Exactive HF mass spectrometer was operated in datadependent acquisition mode with a Top20 strategy corresponding to the selection of the 20 most abundant precursor ions for serial fragmentation. Full-scan mass spectra were acquired from 350 to 1,800 m/z. Only peptides with 2 or 3 positive charges were selected for fragmentation with a dynamic exclusion time of 10 sec and an isolation window of 1.6 m/z.

The LTQ Orbitrap XL was operated with the same parameters but with a Top5 strategy corresponding to the selection of the 5 most abundant precursor ions, an isolation windows of 1 m/z, and a linear 60 min gradient (5% B from 0 to 3 min and 5-50% B from 3 to 63 min).

High-throughput proteotyping of microorganisms

Cross-contamination tests and microorganism screening were performed using a doublebarrel LC-MS/MS system, based on the same principle as that described by Hosp et al. [START_REF] Hosp | A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day[END_REF]. This parallel UHPLC system operating with two analytical columns reduces the idling time of the mass spectrometer during loading of the peptides to the reverse-phase column. Two pairs of PepMap 100 C18 µ-precolumns and PepMap 100 C18 nanoLC columns were mounted in tandem on the same chromatography platform coupled to the LTQ-Orbitrap XL tandem mass spectrometer, allowing elution of peptides from one column in parallel to washing and reconditioning of the other. In this case, peptides (30 ng) were desalted and resolved with a 27-min gradient consisting of 5-40% B from 3 to 30 min, after a 3-min equilibration at 5% B.

Data interpretation

For the Mix3* digests, MS/MS spectra were assigned to peptide sequences using a database comprising the polypeptide sequences from the annotated genomes of B. subtilis subsp. spizizenii ATCC6633, E. coli BL21-Gold (DE3) pLysS AG and S. cerevisiae S288C strains.

The database contains 43,774 polypeptide sequences and 15,766,031 amino acid residues.

The interpretation was performed using Mascot Daemon software version 2.6.1 (Matrix Science) with the following parameters: 5 ppm peptide tolerance, 0.02 Da MS/MS fragment tolerance, 2+ or 3+ peptide charges, a maximum of two missed cleavages, carbamidomethylation of cysteine as fixed modification, oxidation of methionine as variable modification and trypsin as proteolytic enzyme. Microorganism proteotyping was performed by assigning MS/MS spectra against the NCBInr database (downloaded on 01/03/2018) comprising 108,307,546 protein sequences, using Mascot with the same parameters as above except that only one missed cleavage was allowed, and a p-value below 0.05 in homology threshold mode was set for peptide validation. The phylopeptidomics approach for identifying the microorganisms and quantifying their respective biomass contributions was performed as described [START_REF] Pible | Estimating relative biomasses of organisms in microbiota using "phylopeptidomics[END_REF]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017720.

MALDI-TOF mass spectrometry proteotyping

Material (<5 mg) from recent bacterial colonies (< 48h incubation) was transferred into a 1.5 mL tube and solubilized using 300 µL of Milli-Q water, and then with 900 µL of absolute ethanol. Cells were pelleted by centrifugation at 16,000 × g for 2 min. Proteins were extracted by resuspending the cellular pellets in 50 µL of 70 % formic acid, followed by addition of 50 µL of CH3CN, and vortexing. Samples were centrifuged as described here-above. For each sample, one µL of protein supernatant was spotted onto a MTP384 ground steel MALDI plate (Bruker Daltonics), dried, overlaid with 1.5 µL of α-cyno-4-hydroxy-cinnamic acid matrix (saturated α-HCCA matrix was prepared in 50 % acetonitrile / 2.5 % TFA ), and dried. This was done twice for analytical duplicates. MS spectra were recorded using an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 200-Hz Smartbeam laser. Spectra were recorded in the linear, positive mode from 2,000 to 20,000 m/z, cumulating 500-1,000 shots from different positions of the target spot. The Ion source 1, Ion source 2, Lens and the extraction delay time were set at 20.07 kV, 18.60 kV, 7.50 kV, and 200 ns, respectively. Each spectrum was the merged signal from 500-1,000 shots from different positions of the target spot. Calibration was performed externally using the Bruker Bacterial Test Standard (Escherichia coli protein extract including the additional proteins RNase A and myoglobin). Spectra were recorded with the flexAnalysis software (v3.4, Bruker Daltonics) and interpreted with the Biotyper software (v3.1, Bruker Daltonics) and the biotyper spectral database v7 which contains spectra profiles for 7,311 reference microorganisms. The proteotyping confidence is based on a score analysis as recommended by the manufacturer: i) a score > 2.3 was considered highly probable species identification, ii) a score between 2.0 and 2.3 was considered a secure genus identification and probable species identification, iii) a score between 1.7 and 1.999 was considered as a probable genus identification, iv) a score < 1.7 was considered as not a reliable identification.

Results

Performance of Smart2 MBS and MagnaBind for 96-well plate proteotyping

SP3 magnetic-bead proteolysis of proteins requires low volumes of reagents and the resulting digests are ready for direct LC-MS/MS analysis [START_REF] Hayoun | Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry[END_REF]. It is important, however, to ensure that the final digests do not contain any magnetic beads that could disturb peptide injection or liquid chromatography, particularly for the 96-well plate configuration. We evaluated the performance of two commercial neodymium magnetic racks in terms of bead trapping: the Smart2 MBS (Tecan) and the MagnaBind (Thermo Scientific). The former rack has 108 cylindrical magnets arranged above and below each well, with direct contact between the bottom of each well and the magnet. The latter has 24 large magnets, placed in-between four wells. In this case, the magnets are covered by a plastic plate of 0.5 mm thickness, which increases slightly the distance between the magnets and the beads as compared with the Smart2 MBS magnetic rack. The magnetic field for both devices is not given by the suppliers.

The operational performances of the two magnetic racks were evaluated by processing the E. coli lysate. Figure 1 shows how beads are retained at the bottom of each well and whether some beads could be present in the final peptide samples. As shown in Figure 1 (panel A), the two magnetic racks have a different distribution of beads during magnetic retention. The Smart2 MBS magnet traps the SP3 beads in a small spot at the very bottom of the well, whereas the MagnaBind rack beads are concentrated on the wall of the tube but not at the bottom. With regards to the MagnaBind magnet, we observed a partial resuspension of some beads when removing the liquid during the different washing steps, which tended to diminish the amount of trapped material. Also, some beads were recovered in the final peptide sample.

The attraction of the beads on the wall of the tube rather than at the bottom also made a difference when removing the fluid. In the former case, the fluid may scrub the beads upon aspiration, while this may be attenuated when the beads are at the bottom of the well. The presence of beads in the final digests was inspected by optical microscopy. Figure 1B shows the absence of magnetic beads in digests obtained using the Smart2 MBS rack, whereas some beads were observed in the digests using with the MagnaBind rack. Therefore, it is advisable that the use of MagnaBind magnet rack for proteolysis should be followed by a supplementary clean-up step before LC-MS/MS analysis of peptide samples, as beads may damage the chromatography system. This additional cleanup consists of introducing each digest into a new plate and, after magnet retention, re-aspirating the peptide samples. We measured the average peptide digest volume obtained with the two 96-well plate protein digestion formats. Both the Smart2 MBS and MagnaBind racks showed some sample losses, as only 8.02 µL (±4%) and 7.26 µL (±8%) were recovered, respectively, from the initial 10 µL enzymatic solution added. This volume loss can be explained by bead rehydration and evaporation during the enzymatic digestion step. Based on these results, the Smart2 MBS magnet rack is recommended if sensitivity and simplicity are required. Accordingly, the next steps of proteotyping validation described were performed with the Smart2 MBS magnet rack.

A 96-well SP3 approach for high-throughput analysis performs equally well as in-tube format We compared the proteotyping efficiency of the SP3 sample preparation in a 96-well format with an in-tube SP3 digestion using the Mix3* asymmetric mixture of microorganisms containing 94.2% of B. subtilis, 5.7% of E. coli and 0.2% of S. cerevisiae cells. A total of 37,137 (±4%) MS/MS spectra were recorded for the in-tube SP3 with a starting material of 0.34 mg of cellular pellet (wet weight). Most of these spectra (20,567, ±4%) could be attributed to peptide sequences, leading to the identification of 13,311 (±4%) peptides and 1,873 (±1%) proteins validated with at least two different peptide sequences. The peptidespectrum matches (PSMs) found for each sample and the corresponding proteins are listed in Supplementary Tables S1 andS2, respectively. The same proteolysis protocol performed in the 96-well plate format with the Smart2 MBS magnet had no significant impact on digestion efficiency and reproducibility, as it yielded 19,275 (±6%) PSMs, 12,436 (±4%) peptides and 1,715 (±2%) proteins (see Supplementary Tables S3 andS4 for the list of PSMs and proteins, respectively). Likewise, the same ratios of missed tryptic cleavage were found between the in-tube and 96-well platforms, with 79% of peptides without miss-cleavage, 19% with a single miss-cleavage and 2% with two miss-cleavages whatever the method. The detected peptides and identified proteins from the two conditions had similar physicochemical properties. These two conditions share most of their peptidome, with only 16.2 % and 8.7 % of unique peptides specific for in-tube and 96-wells proteolysis, respectively. Figure 2 shows a Venn diagram comparing the number of proteins validated with at least 2 peptides in both digestion conditions. When considering all the proteins identified in the 3 replicates, only 12.2 % and 4.2 % were specific to in-tube and 96-wells SP3, respectively. A total of 183 proteins among the 250 exclusively identified in the in-tube condition are also identified but with only a unique peptide in the 96-wells SP3 sample. Similarly, 70 of the 87 specific proteins presents in the 96-wells dataset are also found in the in-tube results with a unique peptide. As expected, experimental and analytical variability observed at the peptide level influenced the validation of a few number of proteins. However, these results demonstrate that such variability does not significantly change the confidence of the proteotyping analysis. Also, their proteotyping values were equivalent, as the number of peptides assigned to each microorganisms were comparable between the 96-well format (Table S3) and the in-tube format (Table S1): 665 and 791 for S. cerevisiae, 3671 and 4157 for E. coli, and 11542 and 12348 for B. subtilis.

Relative quantities of microorganisms in the Mix3* are conserved

A modified preparation protocol can result in alterations in accuracy or precision in the relative quantification of mixtures of organisms. This can be problematic in studies aiming to monitor simple or complex mixtures of microorganisms in different conditions or over time, for example, clinical, biomimetic, biotechnological or bioremediation microbiota setups. We thus questioned whether the relative quantification of microorganisms was comparable between in-tube-and in-plate-based SP3 protocols. The Mix3* sample is an excellent substrate for estimating alterations in relative quantity as the 3 different microorganisms are strongly heterogeneous in terms of cell wall and membrane structure: gram-positive bacteria, gram-negative bacteria and yeast. The relative quantities of the three organisms were assessed by phylopeptidomics [START_REF] Pible | Estimating relative biomasses of organisms in microbiota using "phylopeptidomics[END_REF] in triplicate and are reported in Table 1. Results showed that the biomass ratio for the three organisms was comparable between in-tube and in-plate SP3 protocols and corresponded relatively well to the protein quantities that can be extracted from the cells.

Range of use of the SP3 96-well plate format for proteotyping

In routine diagnostics, proteotyping of microorganisms can be performed on samples containing various biomass quantities. Therefore, we investigated the application range of the SP3 96-well plate proteotyping method by testing different quantities of E. coli cellular lysate.

The lysate from 1.7 × 10 9 E. coli cells corresponding to 300 µg of protein was diluted to give an equivalent of 5.7 × 10 4 cells (0.01 µg of protein) for the lowest level. Figure 3 shows the bead retention achieved by the Smart2 MBS magnetic rack after the digestion step. We noted that the protein concentration has a direct effect on bead attraction efficiency. The presence of a large amount of protein enhances bead aggregation (for example, compare well A6 to well A3 in Figure 3). By contrast, when the protein quantity was excessive (above 100 µg), a loss of proteins during the washing steps was observed. The resulting peptides for each sample were analyzed by LC-MS/MS and the species present in the sample was identified by MS/MS proteotyping. The number of PSMs assigned to E. coli and the number of taxon-specific peptides are listed in Supplementary Table S5. As shown in Figure 3, an adequate number of species-specific peptides were obtained in all the quantities tested, except for the quantity of 10 ng of protein, equivalent to 5.7 × 10 4 cells. We also noted low values for speciesspecific peptides at 2.8 × 10 5 and 1.7 × 10 9 E. coli cells, with only two species-specific peptides in most replicates. In the latter case, the presence of an excess of proteins disturbed the retention of beads during the process and completion of digestion, as illustrated by the strong decrease of MS/MS spectra (35%) and PSMs (56%). The SP3 96-well plate format is thus adaptable for proteotyping microorganisms in a broad range of material quantity from 2.8 × 10 5 to 1.7 × 10 9 cells.

No cross-contamination occurs for proteotyping in a 96-well plate format

The 96-well plate SP3 format is an attractive solution for high-throughput proteotyping.

However, cross-contamination of unwanted material between wells, due to the addition/removal of reagents or mixing steps, could be disadvantageous as compared with an in-tube protocol where the use of separate tubes limits inter-sample contamination. The possibility of cross-contamination was tested by parallel microplate digestion of two model bacteria, A. baumanii and E. coli, which are phylogenetically distant and thus easily distinguishable. In this case, one species was introduced into a well and the other species was then introduced in all surrounding wells (Figure 4). The reciprocal experiment was also setup and both experiments were performed twice. After plate processing, the resulting 36 digests were subjected to nanoLC-MS/MS and proteotyping identification. The detailed proteotyping data are detailed in Supplementary Table S6. The 36 samples were accurately identified at the species level by 1,026 (±3%) PSMs and 10 (±13%) assigned specific peptides for A. baumanii, and 1,273 (±2%) PSMs and 2 (±20%) assigned specific peptides for E. coli.

The presence of E. coli in A. baumanii samples, or the reverse, was not detected through PSMs or specific peptides. These results demonstrate that sample homogenization, liquid disposal, reagent removal and incubation steps during the protocol do not have any impact on the purity of samples. Thus, the method can be easily applied to a microplate platform without any risk of cross-contamination.

Application of the 96-well plate format for screening isolates by tandem massspectrometry proteotyping

To assess the real-life performance of the 96-well plate SP3 format for proteotyping clinically-relevant microorganisms, we sampled dental plaque and plated microbial isolates on agar. A total of 48 isolates were randomly selected and treated in duplicate with the 96well plate SP3 protocol to produce peptides. The 96 peptide pools were then analyzed by double-barrel chromatography-tandem mass spectrometry to establish whether the samples corresponded to a pure isolate or to a mixture of microorganisms, and to identify them at the most relevant taxonomical level. The proteotyping results with the confident taxonomical level reached for the 96 samples are shown in Figure 5 and the corresponding number of MS/MS spectra, PSMs and specific peptides are listed in Supplementary Table S7.

Considering all the samples, a mean of 3,037 (±15%) MS/MS spectra were recorded for each sample and 1,184 (±22%) peptide sequences were identified. Notably, the same identification result was obtained for each duplicate, demonstrating the good reproducibility of the sample preparation method for a large number of samples. As expected, different taxonomical levels could be ascertained depending on the sample. The 48 isolates were assigned at the species level, with a significant number of species-specific peptides or assigned PSMs. As shown in Figure 5, the identified microorganisms were quite diverse, with Stenotrophomonas maltophilia (8 isolates), Pseudomonas aeruginosa (8 isolates), Staphylococcus aureus (5 isolates), Serratia marcescens (4 isolates) and Lactococcus lactis (3 isolates) being the most represented. Several isolates belonging to various species of Rothia (4 isolates), Neisseria (6 isolates) and Staphylococcus (8 isolates) were also identified. For example, Rothia dentocariosa, Rothia aeria, and Rothia sp. HMSC067H10 (Rothia dentocariosa-like) were classified. Among the 48 isolates, five corresponded to mixtures of two microorganisms: L. lactis and Staphylococcus warneri (sample 2), S. marcescens and Cronobacter dublinensis (sample 16), S. marcescens and S. maltophilia (sample 17), Massilia sp. Leaf139 (Massilia timonae-like) and Curtobacterium flaccumfaciens (sample 25) and P. aeruginosa and Microbacterium sp. Ag1 (sample 30). The tandem mass spectrometry identification thus highlighted the presence of mixtures of bacteria and could precisely identify the most abundant species. S. marcescens was the predominant bacterium of sample 16 analyzed in positions G4 and H4 with the presence of a low amount of C. dublinensis (8% of the total assigned PSMs), and of sample 17 in positions A5 and B5 with some S. maltophilia (21% of the total signal). Sample 30 corresponded mainly to P. aeruginosa with a small contamination of Microbacterium sp. Ag1 (3% of the assigned PSMs). Overall, the optimized approach combining the 96-well plate SP3 preparation, tandem mass spectrometry and phylopeptidomics led to the characterization of 96 samples in 55 hours. Twenty-four of the described samples were analyzed by MALDI-TOF mass spectrometry, after protein extraction in order to maximize the identification (Supplementary Table S8). Only 3 samples were identified with highly probable species score, 7 with a probable species score, 9 with a probable genus score, and 5 could not be identified. The MALDI-TOF results of 10 samples are concordant with MS/MS proteotyping, among which only two bacteria with a highly probable species identification and four with secure genus identification and probable species identification.

Discussion

SP3 paramagnetic beads offer fast purification and proteolysis of proteins [START_REF] Hughes | Ultrasensitive proteome analysis using paramagnetic bead technology[END_REF][START_REF] Hughes | Singlepot, solid-phase-enhanced sample preparation for proteomics experiments[END_REF]. In the present study, we assessed their application for microorganism identification by tandem mass spectrometry proteotyping after optimization, and we found them to be more efficient and quicker than other sample preparation methods [START_REF] Hayoun | Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry[END_REF]. The 96-well plate format allows simultaneous treatment of many samples, resulting in significant time saving. The results indicate a low variability of in-tube and 96-wells SP3 proteolysis, with 83.6 % of shared proteins. An important part of the specific proteins are detected in both conditions but not validated with at least 2 peptides. This rather low experimental variability can be explained by small differences between in-tube and 96-wells procedures in terms of contact area liquid volume recovered after digestion. Furthermore, this format opens the door for fully automatized sample preparation, which is important in the framework of clinical diagnostics of highly virulent pathogens that require minimal handling by operators. We show here that the SP3 96-well plate format is adaptable for proteotyping microorganisms over a broad range of cellular material, from 10 5 to 10 9 cells. Such quantities are easily obtainable by picking a colony from an agar plate or by centrifuging a few milliliters of liquid culture. We did, however, note some difficulties in protein purification for larger quantities of material. Thus, the operator should not overload the wells. A recent publication by Muller et al. [START_REF] Müller | Automated sample preparation with SP3 for low-input clinical proteomics[END_REF] presents an implementation of SP3 using a liquid handling Bravo system (Agilent Technologies) for a fast and automated 96-well format sample preparation for performing proteomics. In terms of protein concentration, our method is conform to the reported observations with capabilities to work with less than 100 ng of proteins. The E. coli cells used in our study are much smaller than HeLa cells. The volume of the former is roughly 1000 smaller than the later. Therefore, for the same input material, a larger number of cells is required: approximately 10 5 E. coli cells are equivalent in terms of proteins to 100 HeLa cells. In addition to the interesting conclusions presented by Muller et al. [START_REF] Müller | Automated sample preparation with SP3 for low-input clinical proteomics[END_REF], our results demonstrates that excess sample may have an impact on the procedure and that the conformation of magnets on the rack may lead to a decrease of protein recovery. As demonstrated here, tandem mass proteotyping with sample preparation performed in 96-well plates does not seem to carry a risk of crosscontamination of samples under standard conditions. Nevertheless, for quality control, we would recommend using specific wells on the 96-well plate for systematic controls with known strains and calibrated bacterial loads. This would allow the assessment of i) correct sample processing and ii) the absence of cross-contamination in each plate. The easy handling and low steps of the 96-well SP3 protocol presented in our publication is adapted for automated system, as already described in the literature. The Smart2 MBS magnetic rack selected for this efficient beads retention is part of a workstation (Tecan) and can be used for automation without any need of protocol adaptation.

Whole-cell MALDI-TOF mass spectrometry has been successfully implemented in clinical laboratories because sample preparation is simple and fast, it does not require heavy cell loads, and there is limited operator-contact with pathogens. The tandem mass proteotyping pipeline presented here, and more specifically the sample preparation in the 96-well format, holds the same qualities. While whole-cell MALDI-TOF mass spectrometry has quickly become a standard approach for identifying human pathogens because of its simplicity to implement and low cost [START_REF] Rodriguez-Sanchez | Review of the impact of MALDI-TOF MS in public health and hospital hygiene[END_REF][START_REF] Singhal | MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis[END_REF], some limitations have been highlighted [START_REF] Grenga | Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns[END_REF]. In particular, several microorganisms are not well identified at the species level; for example, Streptococcus pneumoniae and Streptococcus mitis cannot be easily discriminated with this methodology [START_REF] Varghese | Current challenges in the accurate identification of Streptococcus pneumoniae and its serogroups/serotypes in the vaccine era[END_REF]. Tandem mass spectrometry proteotyping is based on many more experimental features than whole-cell MALDI-TOF mass spectrometry: thousands of peptide masses recorded with high accuracy (error below 5 ppm) versus less than one hundred protein molecular weights with low accuracy (error above 400 ppm), respectively. Thus, the former approach is more discriminant than the latter as confirmed with the comparison carried out in the present study. The MS/MS spectra were recorded using a mass tolerant precursor of 5 ppm, a mass fragment ion of 0,02 Da and an isolation window of 1.6 m/z. These values recommended by Thermo Scientific for measuring MS/MS spectra should be used for interpreting MS/MS proteotyping results. Because of the extra-large database used, appropriate control of the FDR is key for avoiding false-positive identification of microorganisms. Noteworthy, the additional signals recorded by MS/MS could increase the sensitivity of strategies related to high throughput dereplication of isolates [START_REF] Dumolin | Introducing SPeDE: High-Throughput Dereplication and Accurate Determination of Microbial Diversity from Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry Data[END_REF] and could improve discrimination of strains at taxonomic ranks below the species level [START_REF] Sauget | Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?[END_REF]. Therefore, tandem mass spectrometry proteotyping could be used for systematically characterizing clinical isolates as a performant first-line epidemiological tool. The 96-well plate format developed here is perfectly adapted for such an objective.

The lack of reference spectra for most environmental bacteria and uncultivable microorganisms is another drawback of whole-cell MALDI-TOF mass spectrometry [START_REF] Grenga | Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns[END_REF]. As shown here for dental isolates, tandem mass spectrometry proteotyping is applicable to any microorganism and, based on the generalist database, can be used to ascertain the phylogenetic lineage of the isolate. Whole-cell MALDI-TOF mass spectrometry is not reliable for characterizing samples containing multiple microorganisms [START_REF] Karlsson | Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics[END_REF]. Here, we confirmed that tandem mass spectrometry proteotyping can be applied to samples containing several microorganisms; indeed, complex mixtures can be characterized in terms of taxa present, but also in terms of biomass contributions [START_REF] Pible | Estimating relative biomasses of organisms in microbiota using "phylopeptidomics[END_REF][START_REF] Kleiner | Assessing species biomass contributions in microbial communities via metaproteomics[END_REF].

In conclusion, the application of an SP3 approach in a 96-well plate format maintains the performance of our previously developed SP3 in-tube method with the added advantages of simpler and quicker processing of many more samples. The illustrative examples presented here demonstrate the potential of combining SP3 sample preparation and phylopeptidomics analysis for rapid and accurate screening of microorganisms, either as isolates or as mixed samples, including uncharacterized strains. We believe this new workflow will become a powerful strategy for high throughput proteotyping of microorganisms from clinical or environmental samples.

10 (

 10 Type Culture Collection), Escherichia coli BL21(DE3) (pET Expression System 30 kit; Novagen) and Acinetobacter baumanii CIP70.Collection of Institut Pasteur, Paris, France) were grown in brain heart infusion broth (bioMérieux), lysogeny broth (BD Bacto) and tryptic soy broth (bioMérieux), respectively.

Figure 1 .

 1 Figure 1. Comparison of SMART2 MBS (Tecan) and MagnaBind (Thermo Scientific)

Figure 2 .

 2 Figure 2. Venn diagram representing the number of Mix3* proteins identified in In-Tube SP3 and 96-wells SP3 proteolysis (n = 3). This diagram includes only proteins validated with at least 2 peptides.

Figure 3 .

 3 Figure 3. Microplate paramagnetic bead proteolysis and proteotyping results for various

Figure 4 .

 4 Figure 4. Schematic view of Acinetobacter baumanii (orange spots) and Escherichia coli

Figure 5 .

 5 Figure 5. Proteotyping results of isolates from dental plaque. The identified microorganisms are indicated on the corresponding wells from the 96-well plate and their taxonomical classes are distinguished by colors: Gammaproteobacteria (grey), Betaproteobacteria (orange), Bacilli (blue) and Acinetobacteria (green).

Acknowledgements

KH thanks the Région Occitanie for supporting part of his PhD fellowship. This work was funded in part by the French joint ministerial program of R&D against CBRNE threats and the ANR program "Phylopeptidomics" (grant number ANR-17-CE18-0023-01). The authors are indebted to Rosa Maria Gomila Ribas (Universitat de les Illes Balears, Serveis Cientificotècnics, Palma) for performing the MALDI-TOF measurements in a contractual service mode and Sebastian Alberti (Universitat de les Illes Balears) for implementing an excellent technical platform.

Authors contribution statement


Fabrice Anizon

Francis Giraud

Ekaterina S Ivanova

Dmitry N Kaluzhny

Alexander A Shtil

Federico Cisnetti

Pascale Moreau

Synthesis and biological activities of new pyrrolocarbazole-imidazobenzimidazole conjugates

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Among new anticancer therapies, the development of protein kinase inhibitors has emerged as a very efficient strategy, with more than forty drugs that have reached the market to date. As part of our program dedicated to the design and synthesis of new heteroaromatic compounds with promising biological activities, we identified 1,10-dihydropyrrolo[2,3-a]carbazole-3carbaldehyde as a potent and selective inhibitor of the Pim protein kinase family. This compound exhibited in vitro and in vivo anticancer activities, with a significant decrease of the volume and metastatic capacity of PC3 prostate cancer xenografts in mice (compound A, Fig. 1). [1][2][3] N-Heterocyclic carbenes, well-known as ubiquitous ligands in catalysis, [START_REF] Díez-González | N-Heterocyclic Carbenes: From Laboratory Curiosities to Efficient Synthetic Tools[END_REF] are very rarely used as reactants in synthesis. [START_REF] Lake | [END_REF] We have developed a new synthetic method to prepare imidazo[1,2-a]benzimidazole derivatives based on a copper-catalyzed N-heterocyclic carbenenitrene cyclization (compounds B, Fig. 1). 6 Imidazo[1,2a]benzimidazole derivatives are known to be active toward different cellular targets and are therefore relevant in cancer therapy. Indeed, compounds containing the imidazo[1,2a]benzimidazole moiety have been reported for their ability to modulate carcinogen metabolizing enzymes (e.g., inhibition of cytochrome P-450 1A (CYP1A), induction of glutathione (GSH) and glutathione S-transferase (GST)). 7 Furthermore, other reported imidazo[1,2-a]benzimidazoles exhibited inhibitory potencies toward various protein kinases, particularly nanomolar activities toward colony-stimulating factor 1 receptor (CSF1R), a receptor tyrosine kinase that emerged as an attractive immunomodulatory drug target in cancer therapy. 8,9 Drug resistance is a problem that frequently appears after a short period when protein kinase inhibitors are used in monotherapies. One way to circumvent this major drawback and enhance the anticancer therapy potency is the use of multi-target drugs directed toward various cellular structures. 10 Therefore we synthesized conjugated compounds assembling both heteroaromatic scaffolds of A and B via an amide function that could be hydrolyzed by cellular peptidase (Fig. 1).

A R T I C L E I N F O A B S T R A C T

The synthesis of coupling partners 1,10-dihydropyrrrolo[2,3a]carbazole-3-carbaldehyde 6 and imidazo[1,2-a]benzimidazole derivatives 11 and 12, as well as conjugates 13 and 14 will be described (Fig. 1). Then, in order to evaluate the impact of the conjugation on the biological activities of this new compound series, in vitro activities of compounds A, 3, 6, 11, 12 and conjugates 13, 14 will be evaluated toward Pim-1, DNA-binding and five human cancer cell lines (leukemia K562, colon adenocarcinoma HCT116, estrogen receptor positive breast cancer MCF-7, triple negative breast cancer MDA-MB-231 and SCOV ovarian cancer)

Results and Discussion

Chemistry

First, we attempted to prepare the carboxylic acid derivative 6 either from primary alcohols 2 or 3. Compound 1 1 was reacted with 1,4-dibromobutane in order to prepare the known Nsubstituted 4-bromobutyl intermediate. 11 Chromatography on silica gel was performed to remove 1,4-dibromobutane before substitution of the bromine atom by a hydroxyl group in DMSO/H2O in the presence of K2CO3. 12 Partial cleavage of the benzenesulfonyl group was also observed at this step. Therefore, final deprotection of the pyrrolo[2,3-a]carbazole scaffold was performed with NaOH in MeOH/H2O, leading to compound 2 in 44% overall yield. The next step was the formylation of the heteroaromatic part of 2 using standard conditions. 13 However, the alcohol function was also partially formylated. Accordingly, an extra step was performed to hydrolyze the formate intermediate in THF/H2O in the presence of NaOH 14 to give compound 3 (Scheme 1). Unfortunately, despite many attempts using various oxidizing conditions, we were unable to obtain the expected carboxylic acid from 2 or 3. However, as compound 3 constituted a new representative of this interesting pyrrolocarbazole series, its biological activity was also studied.

Scheme 1. Synthesis of the key intermediate 6.

To succeed in preparing compound 6, we used another synthetic pathway consisting first of alkylation of 1 with ethyl 4bromobutanoate in the presence of cesium carbonate and tetrabutylammonium iodide. 15 Next, deprotection of the N-10 position was performed in the presence of magnesium and ammonium chloride in ethanol according to Rosenberg and coworkers. 16 Lastly, a formylation step followed by a basic hydrolysis afforded the carboxylic acid 6 (Scheme 1).

Imidazo[1,2-a]benzimidazole derivatives 11 and 12 were prepared in two steps from previously reported 1-(2azidophenyl)-1H-imidazoles 7 and 8 (Scheme 2). 6 The introduction of the amine chain was performed by alkylation with bromoethylammonium bromide. 17 This procedure delivered an intractable mixture of N-(2-azidophenyl)imidazolium salts 9/10 and protonated starting materials. It was advantageous to perform the cyclization reaction on this crude mixture in the presence of 0.1 eq. copper(I) in aqueous ammonia. Indeed, the cyclization reaction required the formation of an intermediate copper-NHC complex 18 which was possible only from 9/10 and not from protonated 7/8. The latter were easily deprotonated during the last step. Separation of the final mixture relied on the marked difference of physicochemical properties between 7/8 and 11/12. Scheme 2. Synthesis of compounds 11 and 12.

Having prepared all components, the final step was amide bond formation performed in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIPEA). Compound 13 and its trifluoromethylated analogue 14 were obtained in 47% and 50% yield, respectively (Scheme 3). Scheme 3. Synthesis of conjugates 13 and 14.

Biological evaluation

Compounds A, 3, 6, 11, 12, 13 and 14 were evaluated for their Pim-1 kinase inhibitory potencies. The results are expressed as % of residual kinase activities at 10 µM or 1 µM compound concentration. IC50 values were determined only when the % of residual kinase activity was ≤ 25% at 1 µM. As shown in Table 1, the most active Pim-1 inhibitors were pyrrolocarbazoles 3, 6 and conjugates 13, 14 with (sub)micromolar potencies whereas imidazobenzimidazoles 11, 12 did not exhibit any activity.

As planar compounds tend to intercalate in DNA, it is important to verify whether DNA can be an alternative target for these compounds. We tested compounds A, 3, 6, 11, 12, 13 and 14 for their ability to interact with DNA. Drug-DNA complex formation was studied using several approaches. During DNA titration (data not shown), even in the presence of the highest DNA concentration (500 µM base pairs) no significant change in intrinsic compound fluorescence was observed. Alternatively, binding to DNA was also investigated by displacement of the reference intercalating dye ethidium bromide (EtBr). We found that, at concentrations up to 100 μM, no displacement of EtBr was detectable.

Finally, the antiproliferative activities toward 5 human cancer cell lines (chronic myelogenous leukemia K562, colon adenocarcinoma HCT116, estrogen receptor positive breast carcinoma MCF-7, triple negative breast carcinoma MDA-MB-231 and SCOV ovarian carcinoma) were determined to assess the effect of conjugation. The most potent derivatives were conjugates 13 and 14 that demonstrated micromolar potencies toward all tested cell lines while others where either inactive (6, 11, 12) or cytotoxic (IC50 value < 20 µM) only toward some cell lines (A toward MCF-7/SCOV3). Residual kinase activities (% ± SD) and cytotoxicity values (IC50, µM) in brackets. Assays were performed at the International Centre for Kinase Profiling (Dundee, UK). 19 IC50 values of compound A against Pim-1 has been previously reported. 1 clogP values calculated using DataWarrior software. 20 While compounds 6 and 13 exhibited comparable Pim-1 inhibitory potencies, the conjugate 13 was more active toward the cell lines tested. On another hand, 13 and its trifluoromethylated analogue 14 exhibited a cytotoxic potency in the same range, whereas 14 showed milder Pim-1 inhibition. These results demonstrated that Pim-1 inhibition does not correlate with the antiproliferative activities of the tested compounds. Nevertheless, conjugation of pyrrolocarbazole and imidazobenzimidazole moieties slightly enhanced the cytotoxicity compared to the derived compounds. This could be due to an increased intracellular penetration of conjugates 13, 14 bearing an imidazobenzimidazole moiety compared to pyrrolocarbazole 6 (see calculated logP values, Table 1) and/or the involvement of other cellular target(s) important for the cytotoxicity of 13 and 14.

Conclusion

New pyrrolocarbazoles (3, 6), imidazobenzimidazoles (11, 12) as well as pyrrolocarbazole-imidazobenzimidazole conjugates (13, 14) have been prepared in reasonable to good yields. Compounds 3, 6 and 13 exhibited sub-micromolar Pim-1 inhibitory potencies. In addition, conjugates 13 and 14 showed interesting antiproliferative activities in the micromolar range. However, Pim-1 inhibition does not seem to be directly correlated with the antiproliferative activities of these compounds. Nevertheless, the cytotoxicity could be enhanced by conjugation of pyrrolocarbazole and imidazobenzimidazole moieties.

Graphical Abstract

Synthesis and biological activities of new pyrrolocarbazole-imidazobenzimidazole conjugates

Fabrice Anizon, Francis Giraud, Ekaterina S. Ivanova, Dmitry N. Kaluzhny, Alexander A. Shtil, Federico Cisnetti, Pascale Moreau Leave this area blank for abstract info.

Figure 1 .

 1 Figure 1. Chemical structures of lead 1,10-dihydropyrrolo[2.3a]carbazole-3-carbaldehyde (A), imidazo[1,2-a]benzimidazole derivatives (B) and conjugates 13 and 14. In vitro Pim 1-3 inhibitory potencies and cytotoxicities are shown for compound A.

Table 1 .

 1 Kinase inhibitory potencies and antiproliferative activities of reference compound A and new analogues 3

, 6, 11, 12, 13 and 14.

							Antiproliferative activity
			Kinase inhibitory potency
							IC50, µM
	Cpd	clogP
			Pim-1
					K562	HCT116	MCF-7	MDA-MB-231	SCOV3
			10 µM	1 µM
	A	-	(0.12) 1		> 50	33	18	>50	16
	3	-	1.2 ± 0.4	6 ± 2	40	46	27	40	> 50
			(0.039)
	6	3.0	5 ± 2	23 ± 3	> 50	> 50	> 50	> 50	> 50
			(0.20)
	11	-	120 ± 11	114 ± 3	> 50	> 50	> 50	> 50	> 50
	12	-	119 ± 42	143 ± 7	> 50	> 50	> 50	> 50	> 50
	13	3.8	3 ± 0	12 ± 5	9	12	11	14	20
			(0.21)
	14	4.6	22 ± 2	64 ± 13	10	10	14	11	18

Acknowledgments

ESI and AAS are grateful to Blokhin National Medical Research Center (project No. АААА-А18-118022890086-9) for financial support of part of this work. Aurélie Job is acknowledged for HPLC analyses.