Takaaki Tomai
email: takaaki.tomai.e6@tohoku.ac.jp

Elsa Weiss-Hortala

Lin Che

Lin Chen

Supercritical Fluids Technologies for Sustainable Development Goals

Preface

Supercritical fluids technologies for sustainable development goals

This special issue aims to highlight that supercritical fluids technologies can provide promising alternatives to achieve the 17 Sustainable Development Goals (SDGs).

The SDGs were adopted by the United Nations in 2015 as a universal call to action to end poverty, protect the planet, and ensure that by 2030 all people enjoy peace and prosperity. Goal 17. Partnerships for the goals. Nowadays, towards the achievement of SDGs, many countries are setting their national targets and various efforts have been performed in industry, academia and governments. Continuous research and development is needed because it is difficult to achieve the SDGs with the technologies currently in practical use.

Supercritical fluids and related technologies have high potential to contribute to those matters, such as energy, food, health, industry, and environmental issues. For example, related to Goal 7, 9 and 12, supercritical fluid processes and materials synthesized by supercritical fluid processes enable energy-saving chemical processes. Related to Goal 6, 9, 15, supercritical extraction can remediate the contaminated soil. Related to Goal 6, 12 and 14, supercritical water treatment can contribute to the oxidative decomposition of the hazardous waste and to the conversion of biomass and waste plastics into valuable chemicals or chemical raw materials.

In this issue, recent researches on supercritical fluid technologies, targeted on a sustainable society, are presented. The contributions were all peer reviewed and revised according to the standard procedures of the Journal of Supercritical Fluids.

We would like to thank all the researchers that have contributed their efforts to make this special issue possible. Their contributions will help for the development of supercritical fluids technologies in various engineering fields concerning the SDGs and for their implementation in industry.

Takaaki Tomai * Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku

University, Japan

Elsa Weiss-Hortala a,b a RAPSODEE Center, CNRS -UMR 5302, IMT Mines Albi, France b IMRAM, Tohoku University, Japan


Keywords:

Correction data None IMU Installation Lever Arms & Mounting Angles Reference to IMU lever arm [m] -0.013 0.108 -0.007 Reference to IMU mounting angles [deg] 0.000 0.000 0.000 Reference to Primary GNSS lever arm [m] 0.

Objectif et contexte

La mission d'acquisition de données Lidar par drone du 08/03/2022 a été réalisée sur la commune de Terre-de-Bancalié (81), autour du site de La Roque. L'objectif était de réaliser le relevé tridimensionnel du sous-bois dans le but de rechercher des anomalies d'intérêt archéologique autour des vestiges du site castral médiéval.

Conditions de vol

Compte-tenu des contraintes de maintien du drone à vue et du caractère très accidenté et abrupt du versant à documenté, 3 vols ont été réalisés : le premier au départ du fond de vallon, le deuxième depuis le site castral, et le dernier depuis le rebord de plateau. Ces 3 vols ont été réalisés à 50m de hauteur au-dessus du sol à une vitesse moyenne de 4m/s. La couverture obtenue est le meilleur compromis entre la surface à couvrir dans le respect des règles d'usage du drone et la densité minimale des points sols permettant une détection archéologique.

Traitement des nuages de points

Correction des imprécisions GPS

Les données issues de ces vols ont été corrigées des imprécisions GPS en utilisant les données fournies par les bornes du Réseau Géodésique Permanent de l'IGN (disponibles gratuitement sur le site www.rgp.ign.fr) et au logiciel PosPac de Applanix 1 (cf. rapports de correction en annexe).

Extraction des points sols

Après plusieurs tests réalisés en utilisant différents algorithmes et logiciels, c'est finalement le filtrage obtenu avec l'outil d'extraction du terrain de 3D-Reshaper 2 qui s'est révélé le plus satisfaisant. Un premier filtrage « agressif » utilisant un réglage des pentes moyennes de 45 % et une grille d'extraction de 50 cm a été testé, mais c'est finalement un filtrage « adapté » au caractère très accidenté du secteur, utilisant des pentes de 80 % et la même grille d'extraction de 50 cm qui permet la meilleure préservation des infractuaosités rocheuses, mais aussi une part importante de végétation basse qui n'a pas permis au faisceau laser d'atteindre le sol (Figure 3). Ce filtrage « adapté » permet de retenir un peu moins de 9M de points, soit environ 30/m². Les densités sont variables en fonction de la distance par rapport au capteur et les variations de végétation (Figure 4). Nous laissons le soin aux responsables thématiques du projets, meilleurs connaisseurs de leur sujet et de leur terrain, le soin d'interpréter finement ces résultats.

Données livrées avec le présent rapport :

-Nuage de points total au format *.las : 220308_CastelRoc_merge.las -Nuage de points sols filtrés avec le logiciel 3D Reshaper au format *.las : 3Dreshape_sol_80pc.las -Modèle Numérique de Terrain à la résolution de 50 cm issus du nuage de points-sols : raster_3Dreshaper_sol_80pc_50cm.tif -Ensemble des visualisations de ce MNT au format *tif et géoréférencées pour intégration SIG.

-Ensemble des cartes produites pour le présent rapport.

General Information Mission Information

Calibrated Installation Parameters

Reference-Primary GNSS Lever Arm

X Reference-Primary GNSS Lever Arm (m) Y Reference-Primary GNSS Lever Arm (m) Z Reference-Primary GNSS Lever Arm (m) Reference-Primary GNSS Lever Arm Figure of Merit

Smoothed IN-Fusion QC Smoothed Estimated Errors, Reference Frame

Accelerometer Bias (micro-g)

X Accelerometer Bias (micro-g) Y Accelerometer Bias (micro-g) Z Accelerometer Bias (micro-g) Accelerometer Scale Error (ppm) X Accelerometer Scale Error (ppm) Y Accelerometer Scale Error (ppm) Z Accelerometer Scale Error (ppm) Gyro Bias (deg/h) X Gyro Bias (deg/h) Y Gyro Bias (deg/h) Z Gyro Bias (deg/h) Gyro Scale Error (ppm) X Gyro Scale Error (ppm) Roll/Pitch Error RMS (arc-min) Heading Error RMS (arc-min)

Smoothed Solution Status

Processing Mode

Number of Satellites

Baseline Length

SBET IAKAR Separation

Project File List

Rover Data Files

Calibrated Installation Parameters

Reference-Primary GNSS Lever Arm

Smoothed Solution Status

Processing Mode

Number of Satellites

Baseline Length

SBET IAKAR Separation

Project File List

Rover Data Files

Calibrated Installation Parameters

Reference-Primary GNSS Lever Arm

Figure 1 -

 1 Figure 1-Terre-de-Bancalié (81), La Roque -mission du 08/03/2022 -Localisation de la zone couverte par les vols Lidar par drone (Fond -IGN et OSM)

3. 1 .

 1 Évaluation des nuages de points bruts Les 3 vols ont été assemblés en un seul nuage de points comprenant un peu moins de 21 M de points pour une surface documentée de 30 ha, soit une densité d'échos (végétation comprise) d'environ 70 points/m².

Figure 2 -

 2 Figure 2-Terre-de-Bancalié (81), La Roque -mission du 08/03/2022 -Nuage de points total

Figure 3 -Figure

 3 Figure 3-Terre-de-Bancalié (81), La Roque -mission du 08/03/2022 -Nuage de points-sol (filtrage agressif à gauche, adapté à droite)

Figure 5 -FigureFigure 7 -FigureFigure 9 -

 579 Figure 5 -Terre-de-Bancalié (81), La Roque -mission du 08/03/2022 -Hillshade -Azimuth 315° -Hauteur 35°( RVT)

Satellite Lock/Elevation L2 Satellite Lock/Elevation GPS L1 SNR GPS L2 SNR GLONASS L1 SNR GLONASS L2 SNR Roll/Pitch Heading North/East Velocity Down Velocity Total Speed Ground Speed Body Acceleration Total Body Acceleration Body Angular Rate Base Station Information

	Project File List Raw Data QC GNSS QC
	Project name Processing date Mission date Mission duration Processing mode GPS Station Rover Data Files File name YS-20220308-084646.T04 Input Files File Name Ephm0670.22g Ephm0670.22n Raw IMU Import QC Summary IMU data input file IMU data check log file IMU Records Processed Termination Status IMU Anomalies Filename Start date End date Duration Data type Receiver manufacturer, model, serial no. Antenna manufacturer, model GNSS QC Statistics Statistics Baseline length [km] Number of GPS SV Number of GLONASS SV Number of QZSS SV Number of BEIDOU SV L1 Station ID Total number of SV Rover Hardware Information albm067z.22o Antenna height [m] PDOP	CastelRoc 2022-03-09 09:58:29 2022-03-08 08:46:52 00:19:07.997 IN-Fusion Single Base LSDA File type T04 Rover Data File type GLONASS Broadcast Ephemeris GPS Broadcast Ephemeris imu_Mission 1.dat imudt_Mission 1.log 229720 Normal 0 LSDA lsda067z.22o 3/8/2022 8:00:00 AM 3/8/2022 4:59:59 PM 08:59:59.000 GNSS Trimble NetR9 AeroAntenna AT1675-120 w/SPKE 5212K83326 Min Max Mean 23.21 23.58 6 9 9 0 5 4 0 0 0 0 0 0 Dome 7 14 12 GNSS SingleBase 0.072 1.58 2.30 1.90
	lsda067z.22o Antenna measurement method QC Solution Gaps Product Serial number IMU type Offset from measured point to APC [m] Solution Type Output Files Latitude Epoch (s) Receiver type Antenna type Filename sbet_Mission 1.out YS_44_Post_Pac.txt Frame Ellipsoidal height [m] Longitude Percentage	APX 15 AVX 210 VER0 174 GNSS SingleBase Bottom of antenna mount 0.00 0.00 S/N001 0.08085 Fixed Float N43°57'05.59919" 1136.00 0.00 59 APX-15v3 AV14 ASCII Export Output ITRF00 SBET Trajectory File 221.02700 File type E2°10'16.98821" 100.00 0.00	No solution 0.00 0.00
	Epoch	1997
	Ellipsoid	WGS84
	Velocity North [mm/y]	0
	Velocity East [mm/y]	0
	Velocity Up [mm/y]	0

Num SVs in solution Forward/Reverse Separation PDOP Estimated Position Accuracy GNSS-Inertial Processor Configuration

	Processing mode	IN-Fusion Single Base
	Stabilized mount	False
	Base station	LSDA
	Processing start time	204412.003 (3/8/2022 8:46:52 AM)
	Processing end time	205560.000 (3/8/2022 9:06:00 AM)
	Initial attitude source	Primary GNSS Track
	IMU Sensor Context	Processing with Onboard IMU
	Reference to IMU lever arm [m]	-0.013	0.108	-0.007
	Reference to IMU mounting angles [deg]	0.000	0.000	0.000
	Reference to Primary GNSS lever arm [m]	0.056	0.195	-0.417
	Reference to Primary GNSS lever arm std dev [m]	0.100	0.100	0.100
	Aircraft to Reference mounting angles [deg]	0.000	0.000	0.000

Satellite Lock/Elevation L2 Satellite Lock/Elevation GPS L1 SNR GPS L2 SNR GLONASS L1 SNR Roll/Pitch Heading North/East Velocity Down Velocity Total Speed Ground Speed Body Acceleration Total Body Acceleration Body Angular Rate GNSS QC GNSS QC Statistics

	Rover Data Summary Raw Data QC
	First raw data file Raw IMU Import QC Summary	YS-20220308-093749.T04
	Last raw data file	YS-20220308-093749.T04
	File name YS-20220308-093749.T04 Input Files File Name Ephm0670.22g Ephm0670.22n Start GPS week Start time End time Start of fine alignment Available subsystems POS Event Input Correction data IMU data input file IMU data check log file IMU Records Processed Termination Status IMU Anomalies Baseline length [km] Number of GPS SV Number of GLONASS SV Number of QZSS SV Number of BEIDOU SV L1 Statistics Total number of SV	File type T04 Rover Data File type GLONASS Broadcast Ephemeris GPS Broadcast Ephemeris 2200 207493.000 (3/8/2022 9:38:13 AM) 208546.000 (3/8/2022 9:55:46 AM) 207616.000 (3/8/2022 9:40:16 AM) Primary GNSS, IMU None None imu_Mission 1.dat imudt_Mission 1.log 210630 Normal 0 Min Max Mean 23.67 23.95 5 9 9 3 5 5 0 0 0 0 0 0 8 14 14
	albm067z.22o IMU Installation Lever Arms & Mounting Angles PDOP	GNSS SingleBase 1.36 4.79	1.52
	lsda067z.22o Reference to IMU lever arm [m] QC Solution Gaps	GNSS SingleBase -0.013 0.108 0.00 0.00	-0.007
	albm067z.22o Reference to IMU mounting angles [deg] Solution Type	GNSS SingleBase 0.000 0.000 Fixed Float	0.000 No solution
	lsda067z.22o Reference to Primary GNSS lever arm [m] Epoch (s)	GNSS SingleBase 0.006 0.218 1023.00 0.00	-0.435 0.00
	Aircraft to Reference mounting angles [deg] Output Files Reference to Primary GNSS lever arm std dev [m] Percentage	0.000 0.020 100.00	0.000 0.00	0.000 0.00
	Filename	File type
	sbet_Mission 1.out	SBET Trajectory File
	49_PostPac.txt	ASCII Export Output

Num SVs in solution Forward/Reverse Separation PDOP Estimated Position Accuracy GNSS-Inertial Processor Configuration

	Processing mode	IN-Fusion Single Base
	Stabilized mount	False
	Base station	ALBM
	Processing start time	207493.011 (3/8/2022 9:38:13 AM)
	Processing end time	208546.000 (3/8/2022 9:55:46 AM)
	Initial attitude source	Primary GNSS Track
	IMU Sensor Context	Processing with Onboard IMU
	Reference to IMU lever arm [m]	-0.013	0.108	-0.007
	Reference to IMU mounting angles [deg]	0.000	0.000	0.000
	Reference to Primary GNSS lever arm [m]	0.055	0.201	-0.393
	Reference to Primary GNSS lever arm std dev [m]	0.100	0.100	0.100
	Aircraft to Reference mounting angles [deg]	0.000	0.000	0.000

Satellite Lock/Elevation L2 Satellite Lock/Elevation GPS L1 SNR GPS L2 SNR GLONASS L1 SNR Roll/Pitch Heading North/East Velocity Down Velocity Total Speed Ground Speed Body Acceleration Total Body Acceleration Body Angular Rate GNSS QC GNSS QC Statistics

	Rover Data Summary Raw Data QC
	First raw data file Raw IMU Import QC Summary	YS-20220308-102844.T04
	Last raw data file	YS-20220308-102844.T04
	File name YS-20220308-102844.T04 Input Files File Name Ephm0670.22g Ephm0670.22n Start GPS week Start time End time Start of fine alignment Available subsystems POS Event Input Correction data IMU data input file IMU data check log file IMU Records Processed Termination Status IMU Anomalies Baseline length [km] Number of GPS SV Number of GLONASS SV Number of QZSS SV Number of BEIDOU SV L1 Statistics Total number of SV	File type T04 Rover Data File type GLONASS Broadcast Ephemeris GPS Broadcast Ephemeris 2200 210548.000 (3/8/2022 10:29:08 AM) 211393.000 (3/8/2022 10:43:13 AM) 210605.600 (3/8/2022 10:30:05 AM) Primary GNSS, IMU None None imu_Mission 1.dat imudt_Mission 1.log 169130 Normal 0 Min Max Mean 23.44 23.75 7 8 8 4 5 5 0 0 0 0 0 0 12 13 13
	albm067z.22o IMU Installation Lever Arms & Mounting Angles PDOP	GNSS SingleBase 1.37 1.53	1.41
	Reference to IMU lever arm [m] QC Solution Gaps Output Files Reference to IMU mounting angles [deg] Solution Type	-0.013 0.00 0.000 Fixed	0.108 0.00 0.000 Float	-0.007 0.000 No solution
	Filename Reference to Primary GNSS lever arm [m] Epoch (s) sbet_Mission 1.out Reference to Primary GNSS lever arm std dev [m] Percentage	0.006 795.00 File type SBET Trajectory File 0.218 0.00 0.020 100.00 0.00	-0.435 0.00 0.00
	44_CastelRoch.txt Aircraft to Reference mounting angles [deg]	ASCII Export Output 0.000 0.000	0.000

Num SVs in solution Forward/Reverse Separation PDOP Estimated Position Accuracy GNSS-Inertial Processor Configuration

	Processing mode	IN-Fusion Single Base
	Stabilized mount	False
	Base station	ALBM
	Processing start time	210548.002 (3/8/2022 10:29:08 AM)
	Processing end time	211393.000 (3/8/2022 10:43:13 AM)
	Initial attitude source	Primary GNSS Track
	IMU Sensor Context	Processing with Onboard IMU
	Reference to IMU lever arm [m]	-0.013	0.108	-0.007
	Reference to IMU mounting angles [deg]	0.000	0.000	0.000
	Reference to Primary GNSS lever arm [m]	0.051	0.202	-0.396
	Reference to Primary GNSS lever arm std dev [m]	0.100	0.100	0.100
	Aircraft to Reference mounting angles [deg]	0.000	0.000	0.000

1https://www.applanix.com/ 2 https://www.axxess-solutions.com/logiciel3dnuagepoint.html

Y Gyro Scale Error (ppm) Z Gyro Scale Error (ppm)

Commune de , site

Compte-rendu

Mission du 08/03/2022 -Terre-

GLONASS L2 SNR

POSPac MMS 8.3 Diagnostic QC Report -report_Mission 1-CastelRoch-49.pdf -05/16/2022 12:16:48 POSPac MMS 8.3 Diagnostic QC Report -report_Mission 1-CastelRoch-44.pdf -05/16/2022 12:14:39

GLONASS L2 SNR


ˇ´1

Jitka S ˇta´fkova´

Jan Mach

Marc Biran

Zdenek Verner

Frederic Bringaud

Jan Tachezy

Mitochondrial pyruvate carrier in Trypanosoma brucei AQ7

Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of T. brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock-out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought. 44 89 T. brucei, these adaptations include changes in mito-90 chondrial morphology, function, and overall metabolic 91 rearrangements reflected by different spectra of meta-92 bolic end products. In BSF, ATP is primarily generated 93 through glycolysis, and pyruvate is the predominant 94 excreted end product of metabolism (Creek et al., 95 2015). In contrast, PCF that live in the midgut of the 96 insect vector where nutrients are scarce, depend on the 97 mitochondrial catabolic pathway for ATP production. 98 Pyruvate is utilized for substrate level phosphorylation, 99 resulting in the production of acetate and ATP. In addi-100 tion, proline and threonine are important carbon sources 101 for these stages when glucose is limited. In glucose-102 depleted media, proline is metabolized to alanine, gluta-103 mate, CO 2 and succinate, whereas the end products of 104 threonine metabolism are acetate and glycine (Linstead 105 et al., 1977; Lamour et al., 2005).

106

; how-114 ever, there is no information regarding the molecular 115 characteristics of pyruvate transporters in mitochondria. 116 Therefore, the aim of the present study was to deter-117 mine whether T. brucei transports pyruvate into the mito-118 chondrion using a putative MPC homolog and address 119 the relative importance of pyruvate and the pyruvate 120 transporter in PCF and BSF trypanosomes.

121 Results

).

135 The tree shows that Tb927.9.3780 and Tb927.7.3520 clus-136 tered with MPC1 and MPC2/3 orthologs, respectively, with 137 high statistical support. Based on this analysis, the corre-138 sponding T. brucei proteins were designated as TbMPC1 139 and TbMPC2. Subsequently, a series of in silico analyses

Introduction

Pyruvate is a central intermediate metabolite involved in 45 many cellular catabolic and anabolic pathways. This 46 compound is the cytosolic product of glycolysis, and in 47 most eukaryotic cell types, pyruvate enters the mito-48 chondria for further oxidation to acetyl-CoA to fuel the 49 tricarboxylic acid cycle. Thus, pyruvate represents an 50 important branching point in cellular metabolism for bal-51 ancing glycolysis and oxidative phosphorylation. The availability of pyruvate in the mitochondrion is 53 determined through a specific carrier located in the inner 54 mitochondrial membrane. The principal biochemical fea-55 tures of mitochondrial pyruvate carrier (MPC) were char-56 acterized in the 1970s. Pyruvate is symported with one 57 proton, and this transport is driven by DpH (Papa et al., 58 1971; Halestrap, 1978). However, the molecular identity 59 of MPC has only recently been revealed, and currently 60 MPCs have been characterized in Saccharomyces cere-61 visiae, Drosophila melanogaster, Homo sapiens (Bricker 62 et al., 2012; Herzig et al., 2012) and Arabidopsis thali-63 ana [START_REF] Li | 1049 NRGA1, a putative mitochondrial pyruvate carrier, medi-1050 ates ABA regulation of guard cell ion channels and1051 drought stress responses in Arabidopsis[END_REF].

64

The mitochondrial pyruvate carrier comprises two 65 small hydrophobic paralogous proteins, MPC1 and 66 MPC2, which are essential and sufficient for the trans-67 port of pyruvate into mitochondria (Bricker et al., 2012; 68 Herzig et al., 2012). In S. cerevisiae, a third paralog, 69 MPC3, exists, sharing 71% amino acid sequence iden-70 tity with MPC2, and MPC3 expression is induced upon 71 growth on nonfermentable carbon sources (Herzig et al., 72 2012; Timon-Gomez et al., 2013; Bender et al., 2015).

73

Structural predictions have revealed 2-3 transmem-74 brane (TM) helices in all MPC homologs (Bricker et al., 75 2012; Herzig et al., 2012). Pfam lists MPC proteins as 76 members of the MtN3-like clan, together with SWEET 77 transporters ('Sugars Will Eventually be Exported Trans- 78 porters') and the PQ-loop protein family (Finn et al., 79 2014). A previous study suggested that the structure of 80 prokaryotic SemiSWEET transporters, members of the 81 same diverse clan, is similar to that of MPC (Vander-82 perre et al., 2014).

83

Trypanosoma brucei is a pathogen of livestock and 84 humans transmitted through tsetse flies in sub-Saharan 85 Africa. The different life-cycle stages of trypanosomes 86 present specific adaptations to their environments. 87 For the bloodstream (BSF) and procyclic (PCF) forms of 88 was performed. The results from MitoProt II and PSORTII, which predict mitochondrial targeting sequences and subcellular protein locations, indicated the mitochondrial localization of both TbMPC1 and TbMPC2 (the calculated probability of mitochondrial import was 0.85 and 0.83, respectively, according to MitoProt II; the PSORTII k-NN prediction was 47.8% mitochondrial for TbMPC1 and 43.5% cytosolic for TbMPC2). TMpred and TMHMM were used to predict the location of putative transmembrane helices in TbMPCs and all eukaryotic MPC homologs described thus far (Fig. 1B). Experimental evidence for the membrane topology of ScMPC1 and ScMPC2 according to [START_REF] Bender | Regulation 934 of mitochondrial pyruvate uptake by alternative pyruvate 935 936 carrier complexes[END_REF] was considered. Similar to other analyzed MPCs, TbMPC1 and TbMPC2 contain two and three transmembrane domains, respectively. Notably, in all MPC sequences, a tryptophan residue is conserved in the putative binding pocket in MPC2 (W89 in TbMPC2), and this amino acid position is substituted with phenylalanine, another aromatic amino acid, in MPC1 (F52 in TbMPC1). The proline forming the hinge region of the SemiSWEET transporter is conserved in all selected MPC2 and most MPC1 homologs, while trypanosomatid MPC1 revealed a proline-alanine substitution (P50 in TbMPC2, A15 in 162 TbMPC1; Fig. 1B).

Based on the results from the in silico analysis, we concluded that the predicted T. brucei MPCs are evolutionary and structurally related to known MPC proteins.

TbMPC1 and TbMPC2 localization

To determine the cellular localization of MPC paralogs in the PCF and BSF T. brucei, we prepared constructs for expression of TbMPC1 and TbMPC2 with a C-terminal epitope tag. We established PCF cell lines expressing both epitope-tagged TbMPC1 and TbMPC2 and an epitope-tagged TbMPC1 BSF cell line. Despite multiple attempts, we were not successful in obtaining a BSF 174 cell line expressing epitope-tagged TbMPC2.

The localization of TbMPC1 and TbMPC2 was investigated using immunofluorescence microscopy and Western blotting of subcellular fractions (Fig. 1C, Supporting Information Fig. S2). In PCF, both TbMPC1 and TbMPC2 were observed in reticular structures that colocalized with mitochondrial MitoTracker staining (Fig. 1C, panels I, II). To prove the presence of TbMPCs in the mitochondrial membrane, we fractionated crude mitochondrial preparations to obtain mitochondrial matrix-enriched and membraneenriched fractions. Western blot analysis confirmed the presence of both TbMPC1 and TbMPC2 only in the membrane fractions (Supporting Information Fig. S2). Mitochondrial membrane localization of TbMPC1 was demonstrated also in BSF (Fig. 1C, panel III and Supporting Information 189 Fig. S2).

Stage:

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Generation of TbMPC1 and TbMPC2 PCF null mutants and pyruvate uptake analysis

Deletion of the tbmpc1 or tbmpc2 gene was accomplished in wild type PCF (strain 427) by two rounds of transformation in which the ORFs were replaced by an antibiotic resistance gene. Deletion of both alleles was confirmed in 196 several clones by PCR, and clones B6 (Dtbmpc1-B6) and 197 2C4 (Dtbmpc2-2C4) were selected for further experiments 198 (Fig. 2A). Growth of both KO clones in glucose-containing 199 F2 SDM-79 was not significantly affected (Fig. 2B). Thus, 200 The role of TbMPC1 and TbMPC2 in pyruvate import to mitochondria of PCF trypanosomes was assessed by measurement of 14 C-pyruvate uptake by mitochondrial fractions of wild type cells and respective null mutants (Dtbmpc1-B6, Dtbmpc2-2C4). Incorporation of radioactivity in mitochondrial preparations from both mutant cell lines was decreased by approximately 60% compared with wild type samples. A similar decrease in pyruvate uptake was observed in wild type mitochondria incubated with UK-5099, an inhibitor of MPC and monocarboxylate transporters [START_REF] Halestrap | The mitochondrial pyruvate carrier.1012 Kinetics and specificity for substrates and inhibitors[END_REF]. On the contrary, UK-5099 did not affect pyruvate uptake by mitochondrial F3 preparations from either mutant cell line (Fig. 3). These results indicate that both TbMPC1 and TbMPC2 are needed for inhibitor-sensitive pyruvate import into mitochondria and confirm that MPC-mediated pyruvate uptake in the mitochondrion is abolished in both mutant cell lines.

Analysis of metabolic end products in Dtbmpc1-B6 and Dtbmpc2-2C4 PCF cell lines

In PCF trypanosomes, phosphoenol pyruvate derived from glycolysis is further metabolized to succinate in the glycosome, or in the form of pyruvate, it enters mitochondria, where it is metabolized to acetate and succinate F4 (Fig. 4). Therefore, we investigated the effect of tbmpc1 and tbmpc2 gene knockouts on the glucose-dependent 228 formation of metabolic end products. In PCF, pyruvate 229 production was increased in both Dtbmpc1-B6 and 230 Dtbmpc2-2C4 cell lines compared with wild type samples, 231 consistent with defective pyruvate transport into the mito-232 chondrion. In addition, a significant decrease in acetate 233 and succinate production was observed in these mutant 234 cell lines (Table 1). Reduced acetate generation in the 235 T1 mitochondrion is consistent with a reduction in mitochon-236 drial pyruvate metabolism. Null mutants also displayed a 237 trend towards diminished lactate production (P 5 0.07). It 238 AQ4 should be noted that pyruvate cannot be reduced to lac-239 tate in T. brucei due to the lack of lactate dehydrogenase 240 activity. Lactate detected in our assays is expected to be a 241 product of detoxification of methylglyoxal which arises 242 spontaneously in the course of glycolysis (Greig et al., 243 2009). Changes in pyruvate, acetate, succinate and lac-244 tate levels in wild type samples incubated with UK-5099 245 showed patterns similar to those observed in mutant cell 246 lines, albeit these effects were less pronounced. As 247 expected, the presence of the inhibitor did not affect ace-248 tate, succinate and lactate levels in mutant cell lines. 249 Together, these results provide further evidence for the 250 involvement of TbMPC1 and TbMPC2 in mitochondrial 251 To confirm these data, the end products of glucose and/ 253 or threonine metabolism in wild type and TbMPC null 254 mutant cell lines were investigated using H-NMR spec-255 trometry, which facilitates a quantitative comparison, as pre-256 viously described (Millerioux et al., 2012; Bringaud et al., 257 2015). PCF were incubated in PBS containing 4 mM 258 [U-13 C]glucose in the presence or absence of 4 mM threo-259 nine prior to 1 H-NMR quantification of the end products 260 excreted from these carbon sources. In this experiment, 261 threonine-derived acetate production was used as a refer-262 ence to estimate the impact of tbmpc gene deletion on 263 pyruvate-dependent acetate production, as threonine in the 264 incubation buffer served as a substrate for the pyruvate-265 independent acetate production by threonine degradation 266 pathway [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF]. The amount of glucose 267 consumed during the incubation was determined under 268 these conditions and was found to be similar in all three cell 269 lines: 3.33 6 0.36, 3.44 6 0. Direct visual comparison of cells incubated with and 273 without threonine, irrespective of the cell line, revealed 274 slightly lower motility in samples without threonine, 275 although the cells in all samples were viable at the end 276 of the incubation. Production of 13 C-enriched acetate, 277 pyruvate and alanine from [U-13 C]glucose and produc-278 tion of nonenriched acetate from threonine were quanti-279 fied for the three cell lines studied (wild type, Dtbmpc1-280 B6 and D tbmpc2-2C4; Table 2). No succinate was 281 T2 2). In the presence of threonine, alanine production from glucose was observed in wild type and mutant cell lines, whereas in the absence of threonine, relatively small amounts of alanine were only detected in TbMPC null 294 mutants.

Fig. 4. Simplified metabolic scheme of T. brucei. A metabolic scheme depicting the fate of glucose (Glc) and the amino acids threonine (Thr), proline (Pro) and glutamine (Gln). Glucose is eventually converted to cytosolic 3-phosphoglycerate (3-PGA; * -in case of procyclic cells, 1,3-bisphosphoglycerate is exported from glycosomes) through glycolysis within glycosomes (G) and further oxidized to phosphoenolpyruvate (PEP) in the cytosol. A part of PEP is transported back to G and metabolized to oxaloacetate (Oxa). Oxa is converted to malate (Mal), a branchpoint intermediate that can be converted to pyruvate (Pyr) or to fumarate (Fum) in the cytosol, leading to the formation of succinate (Suc) in G, or transported to the mitochondrion (M), where this molecule enters the incomplete tricarboxylic acid cycle (TCA) or is converted to Pyr. The remaining PEP is converted to Pyr, which is either excreted or transported through MPC (full circle) into M. In the mitochondrial matrix, Pyr and Thr are metabolized to acetyl CoA (ACoA), which is subsequently catabolized to acetate (Ace). Both Pro and Gln are converted to glutamate (Glu), which enters the TCA cycle upon transamination with Pyr, yielding alanine (Ala) and 2-oxoglutarate (2-oxo). 2-Oxo is metabolized to mitochondrial succinate (Suc) through TCA enzymes in PCF. During the course of glycolysis, most of dihydroxyacetone phosphate (DHAP) is converted to glyceraldehyde phosphate (GAP), while a minor part of DHAP is spontaneously dephosphorylated to methylglyoxal (Mgx). This harmful compound is then detoxified to L-lactate (Lac). The following enzymes are shown: 1 -triose phosphate isomerase; 2 -glyceraldehyde phosphate dehydrogenase; 3 -phosphoglycerate kinase; 4 -phosphoglyceromutase; 5 -enolase; 6 -phosphoenolpyruvate carboxykinase; 7 -malate dehydrogenase; 8 -cytosolic fumarase; 9 -glycosomal fumarate reductase; 10 -mitochondrial fumarase; 11 -mitochondrial fumarate reductase; 12 -mitochondrial malic enzyme; 13 -pyruvate kinase; 14 -pyruvate dehydrogenase; 15 -threonine dehydrogenase; 16 -2-amino-3-ketobutyrate coenzyme A ligase; 17 -acetate:succinate CoA-transferase; 18 -L-proline dehydrogenase; 19 -pyrroline-5-carboxylate dehydrogenase; 20 -glutaminase; 21 -L-alanine aminotransferase; 22 -nonenzymatic phosphate elimination; 23 -methylglyoxal reductase; 24 -lactaldehyde dehydrogenase; 25 -cytosolic malic enzyme.

Table 1. Effect of mpc1 and mpc2 gene deletion, PEPCK downregulation and presence of the pyruvate transporter inhibitor (UK-5099) on the production of metabolic end products in PCF cell lines.

Metabolic end product

Average6 SD [nmol 3 h 2 1 3 mg protein Metabolic end products were determined by HPLC. n 5 3; The detected differences in metabolites are statistically significant as evaluated using the Kruskal-Wallis test with a 5 0.05.

The preference for threonine over glucose for the generation of acetate (approximately 2.6:1) was documented in wild type samples. This ratio was further increased in both null mutants [approximately 7.9:1 and 7.0:1 in Dtbmpc1-B6 and Dtbmpc2-2C4 cells, respectively (Table 2)], consistent with the expected decrease in pyruvate import into the mitochondrion. The NMR analysis showed that acetate production from glucose, through the pyruvate-dependent pathway, was reduced approximately threefold and fivefold in the TbMPC null mutants in the presence and absence of threonine, respectively. Clearly TbMPC is involved in acetate production from pyruvate; however, the significant residual acetate production from glucose implies that an alternative route is used in PCF trypanosomes.

Rearrangement of metabolism in Dtbmpc1-B6 PCF upon knockdown of PEPCK

Glucose-derived malate enters the mitochondrion and serves as a substrate for the mitochondrial malic enzyme (mitME) in a reaction yielding pyruvate [START_REF] Allmann | Cytosolic NADPH homeostasis in 926 glucose-starved procyclic Trypanosoma brucei relies on 927 malic enzyme and the pentose phosphate pathway fed 928 929 by gluconeogenic flux[END_REF]. We suppressed this pathway in PCF wild type and Dtbmpc1-B6 cell lines by RNAi targeting phosphoenolpyruvate carboxykinase (PEPCK) to distinguish the contribution of TbMPC1-dependent pyruvate transport and malatedependent pyruvate production to the formation of acetate. Downregulation of PEPCK expression in RNAi PEPCK and Dtbmpc1-B6/ RNAi PEPCK samples was examined using Western blotting with an anti-PEPCK antibody (Supporting Information Fig. S3). Clones A11 ( RNAi PEPCK) and C5 (Dtbmpc1-B6/ RNAi PEPCK) were selected for further experiments. No PEPCK signal was detected in either clone, allowing us to assume that PEPCK downregulation was efficient. To avoid the appearance of revertants of constitutive RNAi, freshly selected cells were used in the assays. The growth of PCF 427, Dtbmpc1-B6, RNAi PEPCK and Dtbmpc1-B6/ RNAi PEPCK cell lines in standard SDM79 was comparable (Supporting Information Fig. S3). The end products of glucose metabolism were analyzed using HPLC in knockdown, parental and the Dtbmpc2-2C4 cell 334 lines (Table 1). The production of succinate and fumarate 335 was reduced in both RNAi cell lines, reflecting the partial 336 inhibition of the succinate fermentation pathway. Acetate 337 production was decreased in the RNAi PEPCK and 338 Dtbmpc1-B6/ RNAi PEPCK cell lines compared with the 339 parental wild type and Dtbmpc1-B6 cell lines, respectively, 340 as previously described for the Dpepck mutant (Ebikeme 341 et al., 2010). However, relative acetate production was sim-342 ilar in Dtbmpc1-B6, Dtbmpc2-2C4 and Dtbmpc1-B6/ RNAi-343 PEPCK cell lines (29-33% of the excreted end products 344 from glucose metabolism; Table 1). The same relative pro-345 duction of acetate, regardless of PEPCK expression, 346 strongly suggests that the contribution of malate to mito-347 chondrial pyruvate metabolism is not significant. Alterna-348 tively, the activation of a third unknown adaptive route in the 349 350 double mutant cannot be excluded.

351

Analysis of the Dtbmpc1 BSF mutant cell line

The tbmpc1 gene was deleted in the 427 BSF strain. 352 The deletion of both alleles in several clones was con-353 firmed by Southern blot analysis, and clone 2 354 (Dtbmpc1-2) was selected for further experiments (Fig. 355 2C). Knockin BSF strains expressing epitope-tagged 356 TbMPC1 in Dtbmpc1-2 as well as wild type background 357 were generated and the expression of TbMPC1-HA was 358 confirmed by Western blotting (Fig. 2D). Interestingly, 359 the Dtbmpc1-2 BSF cell line did not exhibit any growth 360 defect when cultured in complex HMI-9 medium (Fig. 361 2E), whereas the growth of this mutant was not sup-362 ported in the recently developed Creek's minimal culture 363 medium (CMM) [START_REF] Creek | Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei[END_REF] (Fig. 2F). Growth 364 rates were calculated from data within the linear sec-365 tions of the curves (96-168 h post introduction to 366 CMM), showing that the growth rate of the deletion 367 mutant reached 36% (relative SD 5 1.6%, n 5 3) of the 368 wild type cell line (100%, SD 5 6.2%, n 5 3). Partial 369 complementation of the growth phenotype was observed 370 in the knock-in cell line in Dtbmpc1-2 background (75%, 371 Table 2. NMR determination of metabolic end products from glucose (Glc) and L-threonine (Thr) in PCF with deleted mpc1 and mpc2 genes.

Metabolic end products from different substrates

Average6 SD [nmol 3 h 2 1 3 mg protein SD 5 2.2%, n 5 3; Fig. 2F). Following the observation of Dtbmpc1-2 growth defect in CMM, we checked whether Dtbmpc1-2 showed altered mitochondrial membrane potential. Parental and Dtbmpc1-2 cells were stained using tetramethylrhodamide ethyl ester and analyzed by flow cytometry using a published protocol (see Subrtova et al., 2015, and Supporting Information). No change in the potential was observed in cells cultured in either media (Mann-Whitney U-test 5 4; P 5 1.0; n 5 3 for both HMI-9 and CMM). Next, we attempted to supplement CMM with all individual components of HMI-9 which are missing in CMM (see Supporting Information Table S5 in [START_REF] Creek | Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose[END_REF] at concentrations present in HMI-9. All individual supplements were tested in a pilot screen aimed at identifying a supplement which (i) would restore the growth of Dtbmpc1-2 cell line in CMM and (ii) would not affect the growth of parental T. brucei Lister 427 BSF. The cells were diluted to 2 3 10 4 cells per ml, aliquoted at 2 ml in culture plates and counted for 3 days after inoculation. HMI-9 and CMM were used as controls. No tested component of HMI-9 matched both requirements, leading us to the conclusion that the growth defect of Dtbmpc1-2 in CMM is not caused by the lack of a single missing component present in HMI-9.

Because the in vitro growth of the Dtbmpc1-2 BSF mutant in CMM was affected, we compared the development of infection in mice. All mice infected with wild type parasites died within 5 days of inoculation, whereas only two out of five Dtbmpc1-2-infected mice died within 10 402 days according to Kaplan-Meier survival curves (log F5 403 rank test 5 9.29; P < 0.01; Fig. 5). Mice infected with the wild type strain also showed high parasitemia, with a maximum of 1 3 10 trypanosomes per ml at 72 h post-inoculation of the parasites. In contrast, the parasitemia in mice infected with Dtbmpc1-2 was considerably lower, with a maximum of approximately 2 3 10 trypanosomes per ml at 96 h post-infection, followed by a subsequent decline. Taken together, these data suggest that in BSF T. brucei TbMPC1 plays an important role in 411 412 vivo and under specific conditions in vitro.

To study acetate production from glucose, the 413 Dtbmpc1-2, tbmpc1 -/1 (single allele mutant) and wild type 414 427 BSF trypanosomes were incubated in PBS containing 415 [U-13 C]glucose and threonine for the 1 H-NMR quantifica-416 tion of excreted end products, as previously described 417 (Mazet et al., 2013; Creek et al., 2015). Apart from pyru-418 vate, which represents ~85% of the end products excreted 419 from glucose, alanine, acetate and lactate were detected 420 using 1 H-NMR. Differences in the levels of acetate pro-421 duced from glucose and the ratio of threonine/glucose utili-422 zation for acetate production were observed. Similar to 423 observations in PCF, the rate of glucose-derived acetate 424 production was significantly decreased in the Dtbmpc1-2 425 cell line (4.6-fold) compared to wild type cells. In addition, 426 an intermediate situation was observed for the tbmpc1 -/1 427 cell line with a 1.5-fold reduction of glucose-derived acetate 428 production compared to wild type (Table 3). This effect 429 T3 shifted the ratio of threonine to glucose utilization for ace-430 tate production from approximately 0.8 in wild type cells to 431 1.3 and 3.7 in the tbmpc1 -/1 and Dtbmpc1-2 cell lines, 432 respectively. As observed for the PCF mutants, the 433 results of the NMR analyses are consistent with a role for 434 TbMPC in pyruvate transport to the mitochondria in BSF 435 436 trypanosomes.

437

Discussion

In this study, we assign a pyruvate transport function to 438 two MPC proteins (TbMPC1 and TbMPC2) in the para-439 sitic protist T. brucei. We demonstrated that both MPC 440 subunits localized in the mitochondrial membrane of 441 PCF T. brucei, and we showed the importance of both 442 subunits for pyruvate uptake using null mutant cell lines. 443 We also addressed the adaptations of metabolic fluxes 444 in MPC deletion mutants in both PCF and BSF trypano-445 somes and investigated the phenotype of BSF tbmpc1 446 447 null mutants in vivo in mouse infections.

In the genome of T. brucei, we identified two MPC paral-448 ogs. MPC proteins are members of the MtN3-like clan, 449 bearing structural similarity to bacterial SemiSWEET pro-450 teins that mediate sugar transport (Vanderperre et al., 451 2014). Whereas the SemiSWEET transporter functions as 452 a symmetrical dimer of triple-helix units, the pyruvate car-453 rier is asymmetrical, comprising two TM helices of MPC1 454 and three TM helices of MPC2 [START_REF] Bender | Regulation 934 of mitochondrial pyruvate uptake by alternative pyruvate 935 936 carrier complexes[END_REF]. We 455 analyzed the primary sequences of MPCs described thus 456 far and TbMPCs, focusing on conserved amino acids impli-457 cated in SemiSWEET transport function. In all analyzed 458 MPC proteins, a conserved tryptophan typical of the Semi-459 SWEET binding pocket is present in MPC2 and substituted 460 for phenylalanine in MPC1 proteins. However, it is unlikely 461 that this substitution affects the transport function of MPCs, as a corresponding tryptophan to phenylalanine mutation in the SemiSWEET transporter did not affect sucrose uptake through SemiSWEET proteins in a liposome assay [START_REF] Lee | Structural basis for the facilitative1046 diffusion mechanism by SemiSWEET transporter[END_REF]. The different number of transmembrane helices in SemiSWEET and MPC transporters results in differences in the binding pocket, which can explain the specificity of the transported substrates (sucrose uniport vs. pyruvate-proton symport). A rather surprising finding concerns the expected hinge region of TbMPC1. All other analyzed MPC1 subunits presented a conserved proline in the second TM helix, consistent with [START_REF] Lee | Structural basis for the facilitative1046 diffusion mechanism by SemiSWEET transporter[END_REF], which serves as an important molecular hinge for the binder clip-like transition between inward-open and outward-open states of the transporter in SemiSWEET proteins and proteins from the PQ-loop family. A proline-to-alanine substitution in SemiSWEET at this site strongly diminishes sucrose transport [START_REF] Lee | Structural basis for the facilitative1046 diffusion mechanism by SemiSWEET transporter[END_REF]. The same substitution is present in TbMPC1. We propose that for the opening of TbMPC pore, it is sufficient for proline-induced intramolecular conformational changes to occur in only one protomer. Both TbMPC1 and TbMPC2 proteins were localized to the mitochondria of PCF trypanosomes by indirect immunofluorescence, and the expression of both proteins was detected in mitochondrial membrane fractions. This result is consistent with previous finding of these proteins in the PCF mitochondrial proteome [START_REF] Panigrahi | A comprehensive analysis of Trypanosoma brucei mitochondrial proteome[END_REF]. The mitochondrial localization of TbMPC1 was also determined in BSF. No staining for TbMPC1 was detected at the BSF plasma membrane, precluding the involvement of MPC in the export of pyruvate as a metabolic end product. It has recently been reported that pyruvate export is mediated through distinct TbPT transporters [START_REF] Sanchez | Molecular identification and characterization of an essential pyruvate transporter from Trypanosoma brucei[END_REF]. Although we were unable to express TbMPC2 in BSF, the localization of TbMPC2 in the BSF mitochondrion is conceivable based on the localization in PCF and the reported association of MPC1 with MPC2 in other species [START_REF] Bricker | A mitochondrial pyruvate car-938 rier required for pyruvate uptake in yeast, Drosophila, 939 940 and humans[END_REF][START_REF] Herzig | Identification1020 and functional expression of the mitochondrial pyruvate1021 1022 carrier[END_REF].

The essential function of both TbMPC1 and TbMPC2 502 for mitochondrial pyruvate transport was directly demon-503 strated by monitoring 14 C-pyruvate uptake in mitochon-504 dria isolated from PCF wild type and TbMPC null 505 mutants. TbMPC-dependent pyruvate import accounted 506 for the total inhibitor-sensitive import, consistent with 507 observations in S. cerevisiae [START_REF] Herzig | Identification1020 and functional expression of the mitochondrial pyruvate1021 1022 carrier[END_REF]. Func-508 tional transport complexes were not detected in either 509 Dtbmpc1-B6 or Dtbmpc2-2C4 cell lines, consistent with 510 available data from yeast (Herzig et al., 2012; Bender 511 512 et al., 2015).

The analysis of the metabolic end products in cell 513 lines lacking either TbMPC subunit strengthened the 514 notion that TbMPCs represent pyruvate transporters in 515 PCF and BSF trypanosomes. Specifically, the decreased 516 production of glucose-derived acetate in both BSF and 517 PCF lacking one tbmpc gene most likely reflects a 518 decreased intramitochondrial pyruvate concentration. 519 However, some glucose-derived acetate is still produced 520 in both PCF and BSF mutant cell lines, while acetate 521 production from glucose is abolished in both BSF and 522 PCF cell lines upon knock down of the subunit E2 of 523 pyruvate dehydrogenase ( RNAi PDH-E2) (Mazet et al., 524 2013; Millerioux et al., 2013). This finding supports the 525 view that an alternative route is used in both trypano-526 some stages to produce/import glucose-derived pyru-527 vate in the mitochondrion. According to the current 528 model of PCF central metabolism, a significant part of 529 the flux from glucose diverted to the glycosome for 530 malate production is exchanged with the mitochondrion 531 to produce pyruvate through mitME inside the mito-532 chondrion [START_REF] Allmann | Cytosolic NADPH homeostasis in 926 glucose-starved procyclic Trypanosoma brucei relies on 927 malic enzyme and the pentose phosphate pathway fed 928 929 by gluconeogenic flux[END_REF]. This hypothesis is con-533 sistent with the observed decrease in succinate 534 production based on HPLC analysis in both PCF null 535 mutants, as a higher proportion of malate would be 536 used as a substrate for mitME to compensate for the 537 lack of pyruvate transport through MPC, eventually lead-538 ing to a decrease in succinate levels. To further address 539 this hypothesis, we analyzed metabolic perturbations or 540 adaptations in Dtbmpc1-B6/ RNAi PEPCK and RNAi PEPCK 541 Cell line Table 3. Effect of mpc1 gene deletion (-/-) and single allele mpc1 deletion (-/1) in BSF on the production of glucose-derived metabolic end products using NMR.

Metabolic end products

Average6 SD [nmol 3 h 2 1 3 mg protein n 5 10. The values indicated in bold represent statistically significant differences in the production of a given metabolite as evaluated using the Kruskal-Wallis test, with a 5 0.05.

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PCF cell lines in which the first step of the glycosomal succinate branch is downregulated. The control RNAi- PEPCK cell line showed a 2-fold reduction of acetate production compared to the wild type parasite, consistent with previous analyses of the Dpepck mutant [START_REF] Ebikeme | Ablation of suc-990 cinate production from glucose metabolism in the procy-991 clic trypanosomes induces metabolic switches to the 992 glycerol 3-phosphate/dihydroxyacetone phosphate shut-993 tle and to proline metabolism[END_REF]. A similar reduction of the rate of acetate production was also observed in Dtbmpc1-B6/ RNAi PEPCK compared to Dtbmpc1 (2.5-fold). These data suggest that either the pathway comprising PEPCK, malate dehydrogenase and mitME contributes poorly to pyruvate/acetate production from glucose in the mitochondrion, or a third route is used in the Dtbmpc1-B6/ RNAi PEPCK cell line, that is, MPCindependent mitochondrial pyruvate transport. Indeed, approximately 40% of the total pyruvate uptake was observed in mitochondria treated with UK-5099 and MPC knock-out cell lines, which is in favour of an alternative pyruvate transporter, such as a member of the monocarboxylate transporter family with broader specificity. Monocarboxylate transporters are typically present at the cell surface, but the mitochondrial localization of these proteins has been reported in rats and baker's yeast [START_REF] Nalecz | Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae)[END_REF][START_REF] Butz | MCT1 confirmed in rat striated muscle mitochondria[END_REF]. However, it cannot be excluded that the mitochondrial membrane integrity of digitonin-permeabilized cells was partially affected in our experiments, facilitating the passive diffusion of 14 C-pyruvate into the mitochondrion. Clearly, additional experiments are required to understand how glucose-derived acetate is produced in Dtbmpc mutant cell lines.

The upregulation of TbMPC proteins in PCF was observed in two proteomic studies, with a fivefold upregulation for TbMPC1 and a fourfold upregulation for TbMPC2 according to [START_REF] Butter | Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery[END_REF] and twofold upregulation for TbMPC1 according to [START_REF] Urbaniak | 1142 Comparative SILAC proteomic analysis of Trypanosoma1143 brucei bloodstream and procyclic lifecycle stages[END_REF]. This finding is consistent with a higher proportion of glucose-derived pyruvate converted into acetate in PCF compared to BSF, representing up to 70% and 5% of the end products excreted from glucose metabolism, respectively [START_REF] Greig | A simple, fast, and1009 accurate algorithm to estimate large phylogenies by max-1010 1011 imum likelihood[END_REF]Bringaud et al., 2015). As expected, no growth defect was apparent in either PCF Dtbmpc1-B6 or Dtbmpc2-2C4 cells in vitro, as the acetate production capacity was not impaired, that is, both mutant cell lines still produced acetate from glucose and threonine, the other source of acetate present in the growth medium.

In contrast to PCF trypanosomes, the MPC gene deletion seems to affect BSF metabolism because (i) the growth of the Dtbmpc1-2 BSF cells was not supported in CMM minimal medium compared with the wild type cells, (ii) we were unable to either express or knock out TbMPC2 in BSF and (iii) the Dtbmpc1-2 BSF mutant showed reduced lethality of infection in mice. Although pyruvate is the principal metabolic end product excreted from glucose metabolism in BSF trypanosomes, the 596 diminished pyruvate transport into the mitochondrion 597 strongly affected this parasite, as an 80% reduction in 598 acetate production was observed in the Dtbmpc1-2 599 mutant (Table 3). In the mitochondrion, pyruvate is fur-600 ther oxidized by PDH, generating acetyl-CoA and even-601 tually acetate. It was recently shown that PDH and 602 threonine dehydrogenase (TDH) are synergistically 603 essential for the growth of BSF in rich medium because 604 of acetate production; both RNAi PDH-E2 and Dtdh single 605 mutants remain viable under these conditions. Accord-606 ingly, the RNAi PDH-E2 cell line is lethal in the absence of 607 threonine [START_REF] Greig | A simple, fast, and1009 accurate algorithm to estimate large phylogenies by max-1010 1011 imum likelihood[END_REF]. Interestingly, the growth 608 of the DTbMPC1 BSF mutant in CMM could not be res-609 cued by threonine, suggesting that the observed reduc-610 tion in acetyl-CoA or acetate production is not 611 responsible for Dtbmpc1-2 death. Alternatively, we 612 hypothesize that MPC is necessary in BSF for supplying 613 the substrate for mitochondrial alanine aminotransferase 614 (AAT). It has been suggested that AAT catalyzing the 615 transamination of pyruvate and glutamate into alanine 616 and a-ketoglutarate is essential in both PCF and BSF 617 [START_REF] Spitznagel | Ala-1125 nine aminotransferase of Trypanosoma brucei -a key1126 role in proline metabolism in procyclic life forms[END_REF]. No activity of the downstream 618 enzyme, a-ketoglutarate dehydrogenase, could be 619 detected in bloodstream T. brucei (Sykes and Hajduk, 620 2013), suggesting that the essential function of AAT 621 might not be directly connected to cellular metabolism. 622 In this context, the question of availability of alanine for 623 mitochondrial translation should be addressed: while it 624 is well documented that mitochondrial translation is 625 essential in both procyclic and bloodstream T. brucei 626 [START_REF] Cristodero | Mitochondrial translation is essential in bloodstream forms of Trypanosoma brucei[END_REF], information on amino acid 627 import into the mitochondrion is very limited in T. brucei 628 [START_REF] De Macedo | An atypical mito-1058 chondrial carrier that mediates drug action in Trypano-1059 1060 soma brucei. PLoS Pathog 11: e1004875. metabolism of the bloodstream forms of Trypanosoma1069 Stage: Page: 14 brucei: production of acetate in the mitochondrion is essential for parasite viability[END_REF] and surprisingly scarce in gen-629 630 eral [START_REF] King | Amino acids and mitochondria[END_REF].

In summary, we identified a mitochondrial pyruvate 631 transporter comprising two subunits and described the 632 properties and function of this protein in the metabolism 633 of a human parasite and an important model organism, 634 T. brucei. Furthermore, these data support the recently 635 emerging picture of BSF functioning well beyond glycol-636 ysis, with unexpectedly active mitochondrial metabolic 637 pathways [START_REF] Roldan | Lipoamide dehydrogenase is essential for both bloodstream and procyclic Trypanosoma brucei[END_REF][START_REF] Greig | A simple, fast, and1009 accurate algorithm to estimate large phylogenies by max-1010 1011 imum likelihood[END_REF]Creek 638 639 et al., 2015). 640

Materials and methods

In silico analyses

641

AQ5

Putative mpc1 and mpc2 genes were identified using 642 BLAST in the T. brucei genome database (www.tri-643 trypdb.org) (Aslett et al., 2010). Both genes were 644 aligned to 29 selected MPC homologs obtained by a 645

Stage:

Page: 10 BLAST search using S. cerevisiae MPCs as queries using Muscle 3.8.425 software (default parameters) [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] and trimmed with BMGE 1.12 (-b 1 -m BLOSUM30) [START_REF] Criscuolo | BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments[END_REF]. The protein evolution model was selected using ProtTest 3.2 [START_REF] Darriba | ProtTest 3: fast selection of best-fit models of protein evolution[END_REF]. PhyML 2.2.0 (topology search: best of NNIs and SPRs, initial tree: BioNJ, Substitution model: LG, proportion of invariable sites: fixed (0), gamma distribution parameter: estimated; number of categories: 4; bootstrap replicates: 500) [START_REF] Greig | A simple, fast, and1009 accurate algorithm to estimate large phylogenies by max-1010 1011 imum likelihood[END_REF] and MrBayes 3.2.2 (rate matrix: LG; rate variation: gamma; gamma categories: 4; chain length: 2,000,000; heated chains: 4; heated chain temp: 0.2; burn-in length: 500,000) [START_REF] Huelsenbeck | MRBAYES:1030 Bayesian inference of phylogenetic trees[END_REF] were used to reconstruct the phylogenetic tree. Transmembrane domains were predicted using the TMHMM Server 2.0 [START_REF] Krogh | Predicting transmembrane protein topology with a1038 hidden Markov model: application to complete genomes[END_REF] and TMPred [START_REF] Hofmann | TMbase -a database1027 of membrane spanning proteins segments[END_REF]. MitoProt [START_REF] Claros | Computational method to predict mitochondrially imported proteins and their targeting sequences[END_REF] and PSORTII [START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF] were used to predict subcellular localization.

Cell cultivation

T. brucei PCF strains 427 and 29-13 (expressing TetR and T7RNAP) [START_REF] Wirtz | A1157 tightly regulated inducible expression system for condi-1158 tional gene knock-outs and dominant-negative genetics1159 in Trypanosoma brucei[END_REF] were grown at 278C in SDM-79 medium [START_REF] Brun | Cultivation and in vitro 946 cloning or procyclic culture forms of Trypanosoma brucei 947 Stage: Page: 13 Mitochondrial pyruvate carrier in Trypanosoma brucei 13 AQ1 in a semi-defined medium. Short communication[END_REF] supplemented with 10% (v/v) fetal calf serum. T. brucei BSF 427 and New York Single Marker (SM, expressing TetR and T7RNAP) [START_REF] Wirtz | A1157 tightly regulated inducible expression system for condi-1158 tional gene knock-outs and dominant-negative genetics1159 in Trypanosoma brucei[END_REF] strains of the same species were grown in HMI-9 medium supplemented with 10% (v/v) fetal calf serum [START_REF] Hirumi | Continuous cultivation of1023 Trypanosoma brucei blood stream forms in a medium1024 containing a low concentration of serum protein without1025 1026 feeder cell layers[END_REF] or CMM supplemented with 10% (v/v) fetal calf serum [START_REF] Creek | Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei[END_REF] at 378C in 5% CO 2 . PCF 29-13 T. brucei were grown in the presence of hygromycin (25 lg/ml) and G418 (15 lg/ml), BSF SM cells were grown in the presence of G418 (1.5 lg/ml). The following concentrations were used for additional antibiotics in cultures of PCF cell lines after transformation: 1 lg/ml of puromycin, 2.5 lg/ml of phleomycin and 10 lg/ml of blasticidin. For BSF mutant cell lines, puromycin at 0.1 lg/ml, blasticidin at 5 lg/ml and phleomycin at 2.5 lg/ml were used. A Z2 cell counter (Beckman Coulter, USA) was used to count the growing trypanosome cultures. The cells were maintained at the exponential growth phase (PCF and BSF were diluted daily to 2 3 10 6 and 1 3 10 4 , respectively), and cumulative cell numbers were calculated.

Generation of mutant cell lines

Tagged TbMPC1 and TbMPC2 were expressed in PCF 29-13 and BSF SM trypanosomes. The entire tbmpc1 ORF was PCR-amplified from PCF Tb427 gDNA using the appropriate primers (see Supporting Information 695 Table S1) and subcloned into plasmid pT7-3V5-PAC 696 [START_REF] Lukes | A novel protein kinase localized to family in Trypanosoma brucei and multiple origins of the1064 lipid droplets is required for droplet biogenesis in trypano-Core I subunit in eukaryotes[END_REF] with a C-terminal V5 tag and a 697 puromycin resistance marker. Plasmid pJH54, with a tri-698 ple HA tag bearing the phleomycin resistance marker 699 (derived from pLEW100; a kind gift from C. Clayton, 700 University of Heidelberg, Germany), was used for 701 702 expression of whole tbmpc2 ORF.

To generate tbmpc1 and tbmpc2 null mutants, the 5 0 -703 and 3 0 -flanking regions of tbmpc1 and tbmpc2 ORFs 704 were PCR amplified (see Supporting Information Table 705 S1 for primers) and sequentially inserted into the plas-706 mids pBS-blast and pBS-phleo [START_REF] Ruepp | Survival of Trypanosoma brucei in the tsetse fly is enhanced by the expression of specific forms of procyclin[END_REF], 707 resulting in four plasmids in which the genes encoding 708 blasticidin deaminase or bleomycin binding protein were 709 flanked by 5 0 and 3 0 UTRs of TbMPC1 or TbMPC2. Two 710 rounds of transformation and selection were required to 711 712 obtain Dtbmpc1 and Dtbmpc2 clonal cell lines.

Knock-in BSF strains expressing TbMPC1-HA in 713 Dtbmpc1-2 as well as wild type background were gener-714 ated using the plasmid pHD1034 [START_REF] Quijada | Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements[END_REF]. 715 Puromycin was used to select stable transformants and the 716 ectopic expression of tagged TbMPC1 driven by an rRNA 717 promoter was checked by Western blotting with a mouse 718 719 monoclonal anti-HA-tag antibody (Sigma-Aldrich, USA).

To generate cell lines showing the constitutively 720 downregulated expression of PEPCK, a sense-antisense 721 3 0 fragment, comprising the portion of PEPCK 722 (Tb927.2.4210) coding sequence and the beginning of the 723 3 0 UTR of PEPCK, was excised from an existing vector 724 [START_REF] Coustou | Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei[END_REF] and inserted in pHD1034 to generate 725 the PEPCK RNAi vector. Wild type and Dtbmpc1-B6 PCF 726 cell lines were used as parental cell lines for the transfor-727 mations. The downregulation of PEPCK expression in both 728 resulting cell lines was evaluated through Western blotting 729 with an anti-PEPCK antibody (a kind gift from Thomas See-730 731 beck, University of Bern, Switzerland).

Linearized plasmids were electroporated into parental 732 PCF T. brucei using two subsequent pulses (1500 V and 733 1700 V) with a Gene Pulser Xcell (Bio-Rad, USA) (Von-734 druskova et al., 2005), or parental BSF was transfected 735 using an Amaxa Nucleofector (Lonza, Switzerland) 736 [START_REF] Burkard | Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters[END_REF], respectively. The transformed 737 cells were subjected to limiting dilution. The growth of 738 PCF clones was facilitated using parental feeder cells in 739 conditioned medium and a 5% CO 2 atmosphere 740 (pouches with CO 2 gen compact, Oxoid). 741 742

Southern blotting

Genomic DNA was isolated from cultures of BSF T. bru-743 cei 427 and Dtbmpc1-2 strains using the TELT method 744 (Medina-Acosta and Cross, 1993) and digested with 745 BamHI or EcoRV enzymes. Approximately 10 lg of digested gDNA was loaded per well. Gel electrophoresis and Southern blotting was performed using standard procedures [START_REF] Southern | Southern blotting[END_REF]. Digoxigenin-labeled probes for the complete coding sequences of TbMPC1 and blasticidine deaminase were prepared using the PCR DIG Probe Synthesis Kit (Roche) with the primers listed in Supporting Information Table S1. Hybridization was conducted using DIG Easy Hyb buffer at 428C. Washing and blocking buffers and anti-digoxigeninalkaline phosphatase with CSPD as substrate were purchased from Roche. Chemiluminiscent signal was detected on ImageQuant LAS4000 (GE Healthcare).

Fluorescence microscopy

Approximately 2 3 10 6 PCF and BSF trypanosomes were used per slide. The PCF cells were incubated with 0.5 lM Mitotracker Red CMXRos (Life Technologies, USA) for 10 min in SDM-79 at 278C, washed with phosphate-buffered saline (PBS), incubated in SDM-79 media for another 20 min and subsequently washed with PBS. The cells were fixed and permeabilized on the slides using -208C cold methanol for 5 min, followed by 5 min incubation in -208C cold acetone.

BSF trypanosomes were incubated with 25 nM Mitotracker Red CMXRos for 30 min in HMI-9 in 378C, collected by centrifugation and incubated again for 10 min in HMI-9, followed by washing with PBS. The cells were fixed on slides using 3.6% formaldehyde for 15 min at room temperature, washed with PBS and permeabilized using 0.1% Triton X-100 for 10 min.

The slides were incubated in blocking solution (0.25% bovine serum albumin, 0.25% gelatin and 0.05% Tween 20 in PBS) for 1 h at room temperature. Expressed TbMPC1-V5 and TbMPC2-HA were visualized using mouse monoclonal anti-V5-tag and anti-HA-tag antibodies, respectively (both from Sigma-Aldrich, USA), and a secondary donkey anti-mouse Alexa Fluor 488 antibody (Life Technologies, USA). The cells were mounted in Vectashield with DAPI (Vector Laboratories, USA) and observed using an Olympus IX81 microscope. The images were captured using a Hamamatsu Orca-AG digital camera and processed using cell^R imaging software (Olympus, Japan).

Infection of mice

Female 8-week-old Balb/c mice (n 5 5) were intraperitoneally infected with 5 3 10 4 BSF trypanosomes. Prior to inoculation, the cells were harvested in mid-log phase from HMI-9 medium and washed once in PBS. Parasitemia was counted in Diff-Quik-stained (Medion Diagnos-tics, USA) smears prepared from the tail blood of 795 infected mice. Animal handling was approved by the 796 Czech Ministry of Agriculture (53407/ENV/13-2300/630/ 797 13). The acquired data were analyzed by Kaplan-Meier 798 799 survival analysis in MedCalc (MedCalc Software). 800

Cell fractionation

Crude mitochondrial fractions were obtained through 801 digitonin solubilization according to [START_REF] Smid | Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma bru-1120 1121 cei[END_REF]. 802 The integrity of the mitochondria and purity of the frac-803 tions were assessed after measuring activities of the 804 cytosolic enzyme pyruvate kinase and the mitochondrial 805 enzyme threonine dehydrogenase as markers for cyto-806 solic and mitochondrial fractions, respectively. The 807 extent of cross-contamination of the fractions was con-808 sistently below 2%. The mitochondrial membrane and 809 matrix fractions were isolated using digitonin according 810 811 to Mach et al. (2013).

To localize expressed recombinant TbMPC1 and 812 TbMPC2 proteins, individual cell fractions were sepa-813 rated using SDS-PAGE, followed by Western blotting 814 and visualization using mouse monoclonal anti V5-tag 815 and anti HA-tag antibodies, respectively, and 816 peroxidase-conjugated goat anti-mouse IgG (Sigma-817 Aldrich, USA). The purity of fractions was evaluated 818 using antibodies against mitochondrial matrix (HSP60), 819 mitochondrial membrane (porin) and cytosolic (enolase) 820 marker proteins, kind gifts of S.L. Hajduk (University of 821 Georgia, USA), M. Chaudhuri (Meharry Medical College, 822 USA) and P. Michels (Catholic University of Louvain,

Pyruvate uptake

Uptake of radioactively labeled pyruvate was performed 826 with digitonin-solubilized cells. Pyruvate stock solution 827 was prepared after mixing nine volumes of 2 mM cold 828 pyruvate with one volume of 2 mM [2-14 C]-labeled pyru-829 vate (ARC, USA). Following solubilization, the cell pel-830 lets (3.5 mg; equivalent of 5-6 3 10 8 cells) were stored 831 on ice. For pyruvate import, the pellet was resuspended 832 in 200 ll of mannitol buffer, pH 7.4 (650 mM mannitol, 833 50 mM potassium phosphate, 1 mM EGTA, 0.1% BSA, 834 10 mM MgSO4 and 1 mM ATP), and incubated on ice 835 for 5 min. Next, the samples were pelleted and resus-836 pended in 200 ll of mannitol buffer, pH 6.3, containing 837 200 lM UK-5099 [alpha-cyano-beta-(2-phenylindol-3-838 yl)acrylate] or the same volume of DMSO (2 ll) for 2 839 min at 278C. Subsequently, 100 lM pyruvate diluted 840 from the stock solution was added, and the samples 841 were incubated at 278C for 15 min. The reaction was 842 quenched after the addition of 1 ml ice-cold mannitol 843 buffer, pH 7.4, containing 10 mM pyruvate. The permeabilized cells were washed four times in quenching buffer and resuspended in 1 ml AquaLuma (Lumac Systems, USA) for scintillation counting.

Analysis of excreted end products from metabolism of carbon sources

High-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) were used to identify and quantify the end products of glucose or L-threonine metabolism.

HPLC analysis was performed using on a Hi-Plex H column (300 3 7.7 mm, 8 lm) (Polymer laboratories, USA) at 658C and a flow rate of 0.4 ml/min, using 5 mM H 2 SO 4 as eluent. The amount of the metabolite was quantified as absorbance at 205 nm. The system was calibrated by 5-point external calibration curves of different concentrations of metabolites expected to be present in the samples. The samples were prepared using the following method: 10 8 trypanosomes per sample were collected through centrifugation, washed and resuspended in glucose incubation buffer (PBS with 24 mM NaHCO 3 and 10 mM glucose, pH 7.3) with 10 lM UK-5099 or the same volume of DMSO (2 ll) to final volume of 200 ll and incubated for 2 h at 278C. Subsequently, the cells were centrifuged, and the supernatant was filtered through a 0.22-mm filter and 30 ll was loaded onto the HPLC column. The output was visualized and analyzed using Clarity 5 software (DataApex).

NMR analyses of end products excreted from glucose and/or threonine metabolism were performed according to [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF] for PCF and Mazet et al. (2013) for BSF. T. brucei PCF (5 3 10 7 ) or BSF (2.5 3 10 7 ) cells were collected after centrifugation at 1400 g for 10 min, washed once with phosphatebuffered saline (PBS) containing 4 mM glucose (BSF) or no glucose (PCF) and incubated for 6 h at 278C (PCF) or 5 h at 378C (BSF) in 2.5 ml of PBS buffer (pH 7.4) containing 4 mM [U-13 C]glucose in the presence or absence of 4 mM threonine. The integrity of the cells during the incubation was assessed through microscopic observation. The supernatant was collected, and 50 ml of maleate solution in D 2 O (20 mM) was added as an internal reference. 1 H-NMR spectra were performed at 125.77 MHz using a Bruker DPX500 spectrometer equipped with a 5 mm broadband probe head. Measurements were recorded at 258C using the ERETIC method. This method provides an electronically synthesized reference signal. The following acquisition conditions were used: 908 flip angle, 5000 Hz spectral width, 32 K memory size and 9.3 sec total recycle time. Measurements were performed with 256 scans for a total 1 time close to 40 min. Prior to each experiment, the 895 phase of the ERETIC peak was precisely adjusted. 896 Resonances of the obtained spectra were integrated, 897 and the results were expressed relative to ERETIC peak 898 integration. The linear production of end products of 899 metabolism of [U-13 C]-glucose ( 13 C-enriched pyruvate 900 and acetate) throughout the experiment was confirmed 901 by H-NMR quantification of the end products excreted 902 by the wild type trypanosomes incubated for 6 h in PBS 903 904 containing 4 mM [U-13 C]-glucose.

905

  52

Fig

  Fig. 1. Phylogeny and localization of MPC1 and MPC2/3. A. An unrooted phylogenetic tree was reconstructed using 29 selected known and predicted MPC homologs. The sequences branched into two distinct clades containing MPC1 or MPC2. Bootstrap values higher than 50 are shown together with posterior probability. The scale bar shows the number of substitutions per site. The sequence alignment, full species names and accession numbers are shown in Supporting Information Fig. S1. B. Alignments of T. brucei MPC1 and MPC2 protein sequences to corresponding orthologs with experimentally confirmed function. Common predicted transmembrane domains (TMpred) are highlighted by a black line. Boxed amino acids correspond to the selected conserved amino acid residues present in functionally important regions of SemiSWEET transporter [substrate-binding pocket and the PQ 'hinge' region aiding the binder clip-like motion of the transporter (Lee et al., 2015)]. C. Immunofluorescent visualization of T. brucei MPC in PCF (I, II) and BSF (III) cells. Antibodies against V5 and HA tags were used to detect TbMPC1 and TbMPC2 (green) in PCF, respectively. V5-tagged TbMPC1 was detected in BSF. MitoTracker (MitT) was used to visualize mitochondria (red), and DAPI was used to visualize nuclei and kinetoplasts (blue). DIC, differential interference contrast.

Fig. 2 .

 2 Fig. 2. Analysis of the genotype and growth phenotype of Dtbmpc1-B6 and Dtbmpc2-2C4 in PCF cell lines, and Dtbmpc1-2 and knock-in cell lines expressing TbMPC1-HA in BSF. A. PCR analysis of genomic regions flanking tbmpc-coding sequences in wild type and null mutant cell lines. Primers in the mpc coding region and external to the cassette insertion were used (scheme and Supporting Information Table S1). Water instead of DNA was used in the negative control. M: molecular weight marker (*, 500 bp). B.In vitro growth of the wild type PCF strain (open circles), Dtbmpc1-B6 (triangles) and Dtbmpc2-2C4 (diamonds) cell lines. Average values of three independent experiments are shown; relative standard deviation was consistently below 10%. C.Verification of the tbmpc1 deletion in the selected Dtbmpc1 clone 2 using Southern blot analysis. The membranes were probed against TbMPC1 and blasticidin deaminase coding regions. D. Expression of TbMPC1-HA determined by Western blotting in wild type, Dtbmpc1-2, and knock-in BSF cell lines in wild type and Dtbmpc1-2 background. HSP60 expression was visualized as loading control. E, F. Growth of BSF wild type (circles), Dtbmpc1-2 (triangles) and knock-in cell lines (wild type1 TbMPC1-HA, full squares; Dtbmpc1-2 1 TbMPC1-HA, open squares) in HMI-9 (E) or CMM (F). Average values of three independent experiments are shown; relative standard deviation was consistently below 10%.

  1

  63 and 3.89 6 0.11 lmol in wild 270 type, Dtbmpc1-B6 and Dtbmpc2-2C4, respectively [mean 271 272 of 4 or 5 biological replicates6 standard deviation (SD)].

Fig. 3 .

 3 Fig. 3. Effect of the tbmpc1 deletion and MPC inhibition by UK-5099 on pyruvate import into isolated PCF mitochondria. The import of radiolabeled pyruvate was decreased in Dtbmpc1-B6 and Dtbmpc2-2C4 compared to wild type samples. The import of pyruvate in the Dtbmpc1-B6 and Dtbmpc2-2C4 mutants was not affected using the MPC specific inhibitor UK5099, while import was reduced twofold in wild type cells. The error bars indicate standard deviations of three independent experiments.

Fig. 5 .

 5 Fig. 5. Effect of the tbmpc1 deletion in BSF on the viability of infected mice (n 5 5). Survival analysis of wild type-(circles) and Dtbmpc1-2-infected mice (triangles).

  The detected differences in metabolites are statistically significant as evaluated using the Kruskal-Wallis test with a 5 0.05.

	2 1 ] Glucose1 Threonine (Glc 1 Thr) Alanine Glc Acetate Glc Thr 210 6 58 1101 6 97 2816 6 204 1744 6 178 2816 6 204 Total Glc Thr 1738 6 200 270 6 47 Pyruvate Glc 433 6 76 407 6 46 3197 6 243 2415 6 219 3197 6 243 1745 6 36 336 6 59 350 6 54 2443 6 87 2432 6 100 2443 6 87 Page: 7 n 5 5. Stage: Glucose Cell line Pyruvate Alanine Acetate Total Parental 127 6 48 0 1152 6 146 1280 6 193 Dtbmpc1-B6 1064 6 62 67 6 48 252 6 50 1383 6 70 Dtbmpc2-2C4 1037 6 40 108 6 30 207 6 58 1352 6 83 Mitochondrial pyruvate carrier in Trypanosoma brucei 7
	11
	10

Acknowledgements

The authors would like to thank C. Clayton, University of Hei-906 delberg, Germany, and I. Roditi, University of Bern, Switzer-907 land, for plasmids, and T. Seebeck, University of Bern, 908 Switzerland, S.L. Hajduk, University of Georgia, USA, M. 909 Chaudhuri, Meharry Medical College, USA, and P. Michels, 910 Catholic University of Louvain, Belgium, for antibodies. The 911 authors would also like to thank Ivan Hrd´y for consulting 912 throughout the study and Michaela Marcincˇikova´for excellent 913 technical assistance. This work was financially supported 914 through grants from the Biomedicine Centre of the Academy 915 of Sciences and Charles University (CZ.1.05/1.1.00/02.0109) 916 from the European Regional Development Fund, project 917 CZ.1.07/2.3.00/30.0061 from the European Social Fund, the 918 Centre National de la Recherche Scientifique (CNRS), the 919 Universite´de Bordeaux, the Agence Nationale de la Recher-920 che (ANR) through grant ACETOTRYP of the ANR-BLANC-921 2010 and the Laboratoire d'Excellence (LabEx) ParaFrap 922

923

ANR-11-LABX-0024.


Ces Travaux

Mohammad El-Hadj

Nicolas Baghdadi

Samuel Alleaume

gestion des milieux et la biodiversité

INTRODUCTION

OBJECTIF

Les images radars ont montré dans de nombreuses études un fort potentiel par la cartographie des milieux humides. Le CBN Bailleul a récemment réalisé des relevés de végétation ainsi que les cartes de niveaux d'humidité potentiels sur le parc régional Scarpe Escaut. Ces données d'échantillons peuvent contribuer à étudier la capacité des capteurs radars à caractériser les zones humides et plus particulièrement à capter l'hydromorphie des prairies humides. Grâce à sa forte répétitivité d'acquisition, Sentinel 1 permet d'accéder à la dynamique saisonnière des surfaces inondées et à la teneur en eau du sol. Un deuxième radar PALSAR sur ALOS est à tester puisqu'il fonctionne en bande L plus sensible à l'humidité du sol.

Il s'agit donc de tester la détection d'une variable environnementale (hydromorphie) sur une physionomie particulière (prairies). Techniquement, il s'agit d'étudier le potentiel des données radar en bandes C et L pour la cartographie des coefficients d'humidité du sol définis selon la classification d'Ellenberg [1].

ZONE D'ETUDE

Le parc régional Scarpe Escaut est situé au nord de la France dans la région Hauts-de-France.

La superficie du site est de 507 km 2 d'environ (figure 1). Tableau 2 : Nom des cellules utilisées. Enfin, les valeurs de supérieures à 2.0 permettent une séparation presque complète des coefficients i et j.

Date d'acquisition

METHODOLOGIE

RESULTATS

PROFILS TEMPORELS DU SIGNAL RADAR EN BANDE C

La figure 2 (figure 2c). De plus, au cours des jours de l'année 34-52 et 76-88 les valeurs de l'indice de séparabilité pour les paires des coefficients d'humidité du sol 5-9, 6-9, 7-9 sont bonnes (Figure 3c). Par exemple, le jour 88 le signal VV-VH de la classe 9 est inférieur à celui des classes 5, 6, et 7 de 2.3 dB, 2.5 dB, et 3.3 dB, respectivement (figure 2c). De plus, l'indice de séparabilité calculé au jour 88 est de 2.1, 1.9, 1.6 pour les pairs des coefficients 5-9, 6-9, et 7-9, respectivement (figure 3c).

-

PROFIL TEMPOREL DE LA BANDE L

De même, le potentiel de la bande L pour la cartographie des coefficients de l'humidité des sols a été étudié. Les résultats ont montré que :

-La polarisation HV pourra séparer la classe 9 des classes 5, 6, et 7 (figure 6c). En effet, le signal HV au jour 310 (fin septembre) est supérieur de 5.3 dB comparativement aux signaux HV des classes 5 et 6 et de 3.8 dB comparativement au signal HV de la classe 7.

De plus, la valeur de l'indice de séparabilité est d'environ 1.6 pour les paires des coefficients d'humidité du sol 5-9, 6-9, 7-9 (figure 7b).

-

CONCLUSION

Les résultats ont montré que :

-Les capteurs radar aux bandes C et L permettraient de séparer le coefficient d'humidité 9 des coefficients 5, 6, et 7.

-Cette séparation est meilleure en bande C qu'en bande L puisque les indices de séparabilité plus important en bande C qu'en bande L pour les pairs des coefficients 5-9, 6-9, et 7-9.

-Cependant, les résultats associés à la classe 9 restent à prendre avec précaution du fait qu'uniquement deux polygones ont été utilisés pour calculer les informations radar de cette classe.

-Seule la bande C (VV et VH) permettrait de séparer le coefficient 7 des coefficients 5 et 6.

-Enfin, les bandes C et L ne permettent pas de séparer entre les coefficients 5 et 6.

Figure 1 :

 1 Figure 1 : Site d'étude

  Le potentiel des données radar en bandes C et L pour la cartographie des coefficients d'humidité du sol est étudié en analysant le profil temporel du signal radar. Ce profil temporel est élaboré pour chaque bande séparément, mais aussi pour chaque polarisation. Les étapes pour élaborer le profil temporel sont :  Etape 1 : Pour chaque cellule, la moyenne du signal radar en échelle linéaire ( ) a été calculée à partir des images radar. Cette moyenne est convertie ensuite en décibel (dB) [ = 10 × log10 ( )].  Etape 2 : Pour chaque date, les signaux radar (étape 1) de chaque coefficient d'humidité du sol ont été moyennés et l'écart-type a été calculé.  Etape 3 : Enfin, l'évolution temporelle de la moyenne radar (étape 2) a été analysée pour chaque bande. Pour la bande C, le profil temporel a été analysé pour les polarisations VV et VH mais aussi pour la différence entre VV et VH (VV-VH). Pour la bande L, le profil temporel a été élaboré pour les polarisations HH et HV ainsi que pour la différence entre HH et HV (HH-HV). De plus, un indice de séparabilité a été calculé pour chaque paire de classes (coefficients d'humidité du sol). La séparabilité entre les classes i et j [2] est définie par: où et sont respectivement la moyenne et l'écart-type des signaux radar du coefficient d'humidité du sol i. Les valeurs de séparation inférieures à 0.8 signifient que les séparabilités ne sont pas suffisamment importantes pour permettre de distinguer la classe i de la classe j. Dans ce cas, pas plus de 50% des coefficients i et j sont correctement séparés. Pour les valeurs de comprises entre 0.8 et 1.5, la qualité de la séparation entre les coefficients i et j est moyenne.

  figure 2 montre que :

-Figure 2 :Figure 3 :

 23 Figure 2 : Profil temporel du signal radar en bande C (angle d'incidence entre 33° et 36°). (a) polarisation VV, (b) polarisation VH, et (c) polarisation VV -polarisation VH . DOY = Day of

Figure 4 :Figure 5 :

 45 Figure 4 : Profil temporel du signal radar en bande C (angle d'incidence entre 42° et 46°). (a) polarisation VV, (b) polarisation VH, et (c) polarisation VV -polarisation VH. DOY = Day of Year.

  Contrairement à la bande C (VV et VH), la bande L (HH et HV) ne permet pas de séparer la classe 7 des classes 5 et 6. En effet, le comportement du signal radar en bande L pour les classes 5, 6, 7 est similaire (figures 6a et 6b). De plus, l'indice de séparabilité pour les paires de coefficient de l'humidité du sol 5-7 et 6-7 est inférieur à 0.5 (figures 7a et 7b).-Enfin, la bande L ne permet pas de séparer la classe 5 de la classe 6. En effet, les profils temporels de la bande L pour les classes 5 et 6 sont similaires avec des différences qui ne dépassent pas le 0.5 dB en HH, HV et HH-HV (indice de séparabilité ~0.1) (figures 6 et 7).

Figure 6 :Figure 7 :

 67 Figure 6 : Profil temporel du signal radar en bande L (angle d'incidence ~35°). (a) polarisation HH, (b) polarisation HV, et (c) polarisation HH -polarisation HV. DOY = Day of Year.

  Par conséquent, l'utilisation du radar Sentinel 1 (Bande C) semble très pertinente pour capter l'humidité des végétations naturelles ouvertes. Il est important de retenir que le signal radar ne pénètre pas les couverts ligneux importants (buissons ou arbres). Les données Sentinel1 présentent l'avantage d'être disponibles gratuitement sur plusieurs plateformes associées à Copernicus. Une méthode de classification basée sur l'utilisation de la technique d'apprentissage automatique pourrait être envisagée mais reste à développer. Elle pourrait déboucher sur un algorithme permettant de cartographier les indicateurs d'humidité du sol. Bien que de telles approches existent sur la cartographie de l'humidité des zones de culture cela nécessite une adaptation des algorithmes utilisé et une augmentation de l'effort d'échantillonnage pour la calibration et la validation de ces modèles.

pour la


OBJECTIF

Dans le cadre du projet de cartographie des habitats à l'échelle nationale, CarHAB2, il a été proposé de délimiter différents ensembles homogènes du point de vue de la végétation qui les compose. Ce document présente la méthodologie utilisée pour délimiter ces zones floristiquement homogènes, la carte qui en découle ainsi que le recoupement entre les zones identifiées et la zonation en hydro-écorégions. Une interface web a par ailleurs été développée afin de visualiser l'ensemble des résultats obtenus.

DONNÉES

L'établissement de la cartographie voulue repose sur des données géo-localisées de présence des différentes espèces végétales observées en France. Les données de la base SIFLORE ont été utilisées [START_REF] Références Just | SIFlore, a dataset of geographical distribution of vascular plants covering five centuries of knowledge in France: Results of a collaborative project coordinated by the Federation of the National Botanical Conservatories[END_REF]. Ces dernières ont été agrégées par la fédération des conservatoires botaniques nationaux à la maille 5x5 km. Elles ont été fournies au centre Irstea Le tableau 1 nomme les dix espèces les plus fréquentes à l'échelle de la France. Tableau 1 : Dix espèces les plus fréquentes à l'échelle de la France dans la base SIFLORE. Ce score permet ainsi de hiérarchiser pour chaque zone phytogéographique l'ensemble des espèces contributrices, les espèces les plus contributrices ayant un score ρ ij élevé.

MÉTHODOLOGIE

CARTOGRAPHIE OBTENUE

La méthode OSLOM a permis d'identifier vingt ensembles phytogéographiques à l'échelle de la France (figure 4).

LIMITES DE L'APPROCHE

La méthode OSLOM a déjà été utilisée en région méditerranéenne et a permis de délimiter plusieurs zones homogènes du point de vue de la flore qui les compose [START_REF] Lenormand | Biogeographical network analysis of plant species distribution in the Mediterranean region[END_REF]. Ces zones étaient fortement congruentes avec des limites définies par différents botanistes de haute renommée. À l'échelle nationale, cette zone correspond à la zone phytogéographique numéro 20. Ainsi, à partir de données similaires, les zonations obtenues

de

  Montpellier le 23/05/2019. Ces données regroupent la présence de 6650 espèces végétales réparties dans 21612 carreaux 25 km² d'une grille nationale métropolitaine. Pour chaque espèce, la date de première et de dernière observation dans un carreau donné, ainsi que le nombre d'observations entre ces deux dates, est disponible. Seules les données comprises entre 1990 et 2016 ont été traitées. La figure 1 illustre la richesse spécifique disponible dans chacun des 21612 carreaux à l'échelle de la France.

Figure 1 :

 1 Figure 1 : Richesse spécifique par carreau de 25km².

Figure 2 :

 2 Figure 2 : Histogramme de la richesse spécifique par carreau de 25km².

Figure 3 :

 3 Figure 3 : Histogramme des occurrences de chaque d'espèces.

  Afin d'identifier des zones floristiquement homogènes à l'échelle nationale, nous nous basons sur une méthode évaluant la similarité floristique entre les différents carreaux. Une matrice de similarité est ainsi construite à partir de la matrice carreau-espèces fournie. Cette matrice contient un score de similarité pour chaque combinaison de paires de cellules de la base de données, soit (21612*21611) /2 combinaisons.La similarité β sim est évaluée à l'aide de l'indice de[START_REF] Simpson | Mammals and the nature of continents[END_REF] dont la formule est la suivante: β sim =1 -min(b, c)/( a + min(b, c)) Avec a le nombre d'espèces en commun dans une paire donnée de cellules, b le nombre d'espèces uniquement dans la première cellule de la paire et c le nombre d'espèces uniquement présentes dans la seconde cellule de la paire. β sim est compris entre 0 et 1, la valeur maximale correspondant à une composition floristique identique entre deux carreaux et la valeur minimale à une absence totale d'espèces en commun. À partir de cette table de similarité entre paires de cellules, on utilise ensuite une méthode issue de la théorie des graphes pour regrouper les différents carreaux ayant une composition floristique plus proche qu'attendue par hasard. L'approche utilisée correspond à une méthode d'optimisation locale d'une statistique ordonnée (en anglais Order Statistics Local Optimization Method ou OSLOM) (Lancichinetti et al. 2011). Cette méthode itérative détecte des ensembles de paires significativement plus similaires que dans une situation dite nulle où le graphe de similarité entre carreaux est structuré de manière aléatoire. Cette méthode a déjà été employée pour caractériser les ensembles de végétation de la zone Méditerranéenne et a fait l'objet d'une publication dans la revue Ecology and Evolution (Lenormand et al. 2019). La méthode OSLOM permet d'assigner à chacune des cellules du carroyage une zone écologiquement homogène, correspondant à une zone phytogéographique. Une fois ces zones établies, on peut alors identifier quelles espèces végétales contribuent le plus à une zone donnée à l'aide d'un score de contribution. Ce score évalue pour chaque espèce à quel point elle est présente dans des carreaux d'une zone phytogéographique donnée par rapport aux carreaux assignés aux autres zones. La formule suivante est utilisée pour calculer ce score de contribution :Avec n le nombre de cellules définies à l'échelle de la France, i l'espèce focale, j la zone phytogéographique d'intérêt, n i le nombre d'occurrences de l'espèce i à l'échelle de la France, n j le nombre de cellules assignées à la zone j et n ij le nombre d'occurrences de l'espèces i dans la zone j.

Figure 4 :

 4 Figure 4 : Zones floristiquement homogènes identifiées. Chaque couleur correspond à une zone différente.

Figure 5 :

 5 Figure 5 : Superposition des zones floristiquement homogènes identifiées ainsi que des hydroécorégions. Les zones floristiques apparaissent en couleur tandis que les hydro-écorégions sont délimitées en noir.

Figure 7 :

 7 Figure 7 : Onglet avec les différentes régions phytogéographiques.

Figure 8 :

 8 Figure 8 : Espèces contributrices d'une zone phytogéographique.

Figure 9 :

 9 Figure 9 : Hydro-écorégion d'intérêt en surbrillance.

Figure 10 :

 10 Figure 10 : Onglet hydro-écorégion.

  peuvent différer selon les échelles d'étude. Nous proposons donc un deuxième niveau d'analyse visualisable dans l'interface web présentée précédemment. Ce deuxième niveau correspond à une analyse suivant la méthodologie OSLOM au sein des carreaux de chaque ensemble phytogéographique. Ce second niveau d'analyse a permis d'affiner les ensembles obtenus en première instance mais n'aboutit cependant pas à une zonation aussi fine que celle observée par Lenormand et al. (2019) en région Méditerranéenne. La résolution des données, i.e. 25km², est également limitante dans la mesure où elle aboutit à une zonation à une échelle spatiale assez large. L'approche bénéficierait de données à résolutions plus fines et permettrait détailler davantage les ensembles identifiés.Enfin, les approches de délimitation de zones homogènes du point de vue de la flore sont relativement diverses et vont faire l'objet d'une étude plus systématique.