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Major, P., Sendra, K. M., Dean, P., Williams, T., Watson, A. K.,Thwaites, D. T., Embley, T. M., & Hirt, R. P. (2019). A new family ofcell surface located purine transporters in Microsporidia and relatedfungal endoparasites. eLife, 8, [e47037].https://doi.org/10.7554/eLife.47037,https://doi.org/10.7554/eLife.47037
Publisher's PDF, also known as Version of recordLicense (if available):CC BYLink to published version (if available):10.7554/eLife.4703710.7554/eLife.47037
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This is the final published version of the article (version of record). It first appeared online via eLife athttps://doi.org/10.7554/eLife.47037 . Please refer to any applicable terms of use of the publisher.
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Figures and figure supplements
A new family of cell surface located purine transporters in Microsporidia andrelated fungal endoparasites
Peter Major et al
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 1 of 27
RESEARCH ARTICLE
Amphiamblys sp (A8A55_0782)Amphiamblys sp (A8A55_2217)
Nematocida parisii (NEQG_01061)Nematocida sp (NERG_02401)
Nematocida parisii (NEQG_01766)Nematocida sp (NERG_02543)
Edhazardia aedis (EDEG_01832)Antonospora locustae (ALOC_0053)
Anncaliia algerae (H312_00601)Anncaliia algerae (H312_01875)
66
Spraguea lophii (SLOPH_467)
Spraguea lophii (SLOPH_466)
100
100Spraguea lophii (SLOPH_1068)
Spraguea lophii (SLOPH_2261)Trachipleistophora hominis (THOM_1192)
Vavraia culicis (VCUG_01455)Trachipleistophora hominis (THOM_3170)Vavraia culicis (VCUG_01849)
100
100Pseudoloma neurophila (M153_12350001271)
Pseudoloma neurophila (M153_2724000386)Pseudoloma neurophila (M153_2731000541)Pseudoloma neurophila (M153_3302000407)
Vavraia culicis (VCUG_00084)Trachipleistophora hominis (THOM_1681)
40
10099
97
100
98
99
98
85
96
91
100
53
100
99
95
96
Amphiamblys sp (A8A55_2439)Rozella allomycis (O9G_004875)
Rozella allomycis (O9G_000467)Rozella allomycis (O9G_000468)
Rozella allomycis (O9G_003543)Rozella allomycis (O9G_000993)
100
100Amphiamblys sp (A8A55_0330)
Mitosporidium daphniae (DI09_25p180)Mitosporidium daphniae (DI09_30p280)
Mitosporidium daphniae (DI09_202p60)
99
100
Nematocida parisii (NEQG_00533)Nematocida sp (NERG_00050)
Edhazardia aedis (EDEG_00277)Spraguea lophii (SLOPH_2201)
Antonospora locustae (ALOC_0069)Anncaliia algerae (H312_00645)
Anncaliia algerae (H312_02678)Enterocytozoon bieneusi (EBI_22442)
Vittaforma corneae (VICG_02048)
88
Nosema bombycis (NBO_18g0028)Nosema ceranae (NCER_101394)
Nosema apis (NAPIS_ORF00978)100
85
100
Ordospora colligata (M896_121750)Encephalitozoon cuniculi (ECU11_1870)Encephalitozoon intestinalis (Eint_111770)Encephalitozoon hellem (EHEL_111760)Encephalitozoon romalae (EROM_111760)96
6883
Ordospora colligata (M896_121760)Encephalitozoon cuniculi (ECU11_1880)Encephalitozoon romalae (EROM_111770)
Encephalitozoon hellem (EHEL_111770)100Encephalitozoon intestinalis (Eint_111780)
80
100
100
100
57
43
Enterocytozoon bieneusi (EBI_24311)Vittaforma corneae (VICG_01134)
29
100Edhazardia aedis (EDEG_00275)
77
Edhazardia aedis (EDEG_00280)Pseudoloma neurophila (M153_1989000385)
Vavraia culicis (VCUG_01971)Trachipleistophora hominis (THOM_0963)100
100
53
44
100
74
65
79
100
86
54
100
100
85
81
73
97
1
A
B
d daph ( 09 202p60)NeNN matocida pda arisii ii (ii N ( ( EQG_QGQG 00533)NeNN matocida dd sps (NERG_00050)
EdEE hazadd rdrr idd a aedidd sii (EDEG_00277)Sprarr gua ea lophii (SLOPH_2201)
Antonospos ra rr loll cusuu taett (ALOC_0069)Anncaliia algl egg raerr (H312_00645)
Anncaliia algll egg raerr (H312_02678)EnEE terorr cyc tozoo oon bieneusuu i (i EBI_22442)
VittaVittVitt fa off rma cornrr eae (VICG_02048)
88
NoNN sema bombyb cisii (NBO_18g0028)NoNN sema cerarr nae (NC ( ( ER_101394)
Nosema apisii (NAPIS_ORF00978)100
85
100
OrdOrOr odd sps ora crr olligalli tatt (M896_121750)Encephalitozoonitit cuniculi (ECU1i 1_1870)EnEE cephalitolitlit zoo oon intett stiee nalis (Eint_111770)EnEE cepce halitolitlit zoo oon hellellll m (EHEL_1EL 11760)Encephalitozoonitit romalaell (EROM_1117M 60)96
6883
OrdOrOr odd sps ora corr lliglli agg tatt (M896_121760)Encephalitozoonitit cuniculi (ECU1i 1_1880)Encephalitozoonitit romalaell (EROM_1117M 70)
EnEE cepce halitolitlit zoo oon hellellll m (EHEL_111770)100EEEnEE cephalitolitlit zoo oon intett stiee nalis (Eint_111780)
80
100
100
100
57
43
EnEE terorr cyc tozoo oon bieneusuu i (i EBI_24311)VittaVittVitt fa off rma cornrr eae (VICG_01134)
29
100EdEE hazadd rdrr idd a aedidd sii (EDEG_00275)
77
EdhEdEd azardrr idd a aedidd sii (EDEG_00280)PsPP eudodd loll ma neurophilailil (M153_1989000385)
VaVV vrarr ia culicisii (VCUG_01971)TrTT acrr hipii lell isii tophott rarr hominmimi isii (THOM_0963)100
100
53
44
100
74
65
79
100
86
100
A
NeNN matocidadd parisii ii (ii N ( ( EQG_QGQG 01061)NeNN matocida sps (NERG_024RG 01)
NeNN matocida pda arisii ii (ii N ( ( EQG_QGQG 01766)NeNN matocida dd sps (NERG_02543)
EdhEE azardrr idd a aediaedaed sii (EDEG_01832)Antonosps ora rr loll cuscucu taett (ALOC_0053)
Anncaliia algll egg raerr (H312_00601)Anncaliia algl egg raerr (H312_01875)
66
SpSS rarr ga uea lopho ii (SLOPH_467)
SpSS rarr ga uea lopho ii (SLOPH_466)
100
100SpSS rarr ga uea lopho ii (SLOPH_1068)
SpSS rarr ga uea lopo hii (SLOPH_2261)TrTT acrr hipii lell isii toptt horarr hominmimi isii (THOM_1192)
VaVV vrarr ia culicisii (VCUG_01455)TrTT acrr hipii lell isii tott po horarr hominmimi isii (THOM_3170)VaVV vrarr ia culicisii (VCUG_01849)
100
100PsPP eudodd loll ma neuropho ilailil (M153_12350001271)
PsPP eudodd loll ma neurophilailil (M153_2724000386)PsPP eudodd loll ma neurophilailil (M153_2731000541)PsPP eudodd loll ma neurophilailil (M153_3302000407)
VaVV vrarr ia culicisii (VCUG_00084)TrTT acrr hipii lell isii tott po horarr hominmimi isii (THOM_1681)
40
10099
97
100
98
99
98
85
96
91
100
53
100
99
B
Figure 1. Protein maximum-Likelihood phylogeny of MFS transporters from Microsporidia and close relatives. The MFS sequences from the core
Microsporidia (see Corsaro et al., 2016; Galindo et al., 2018) and their close relatives from the genus Amphiamblys, Mitosporidium, and Rozella
Figure 1 continued on next page
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 2 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 1 continued
clustered in two distinct clades based on the rooting inferred from a broader analysis (Figure 1—figure supplements 1 and 2). Microsporidia genomes
encode at least one member of clade A (highlighted with the green box). All Microsporidia sequences (except one - arrowhead) in clade A possess a
distinctive indel (see main text and Figure 1—figure supplement 3). Some sequences from the Rozellomycota including Rozella allomycis,
Mitosporidium daphniae and Amphiamblys sp. also cluster with Microsporidia clade A or clade B sequences (the Microsporidia clade B is highlighted
with the red box). A number of Microsporidia and Rozellomycota species appear to have lost their clade B homologues. Lineage-specific expansions
can be observed for several species in clade A or B. The maximum likelihood phylogeny was inferred with the LG+C60 model in IQ-TREE with ultrafast
bootstrap branch support values (1000 replicates). The scale bar (top right) represents the number of inferred amino acid changes per site.
DOI: https://doi.org/10.7554/eLife.47037.003
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 3 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Tree scale: 1
Fungi
Metazoa
Choanoflagellates
Chlorophyta
Diatoms
Oomycetes
Mucoromycota
Rozellomycota
Microsporidia
Rozellomycota
Microsporidia
Prokaryotes
Clade A
Clade B
Vitrella brassicaformis (Alveolata)
Diatoms (Stramenopiles)
Aureococcus anophagefferens (Heterokontophyta)
Thecamonas trahens (Apusozoa)
Fonticula alba (Nukleariidae)
Trypanosoma (Euglenozoa)
Dictyostelium
Rozellomycota
Glomeromycota
Mucoromycota
BasidiomycotaAscomycota
MucoromycotaMortierellomycota
Chytridiomycota
Beauveria bassiana (Fungi)
100
100
98
88
95
89
100
99
100
Figure 1—figure supplement 1. Maximum likelihood phylogeny of homologues to ThMFS proteins including a broad range of eukaryotes and
prokaryotes. The tree was inferred under the LG+C60 model. The full details, including ultrafast bootstrap support values (1000 replicates), species
names and sequence identifiers are shown in Figure 1—figure supplement 2. The arrow indicates the inferred gene duplication event affecting
Microsporidia and Rozellomycota that gave rise to clade A and B discussed in the text. Ultrafast bootstrap support values (1000 replicates) on key
branches are indicated.
DOI: https://doi.org/10.7554/eLife.47037.004
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 4 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
0.01234567891 01 11 2
Rozella allomycis 528894091
Rhizophagus irregularis 595451823
Lichtheimia corymbifera 661189283
Phycomyces blakesleeanus 1026593887
Rhizopus delemar 384501739
Choanephora cucurbitarum 1043307241
Parasitella parasitica 758370449
Mucor ambiguus 758354207
1001001001001008 58 58 58 58 5
8 48 48 48 48 49 89 89 89 89 8
5 85 85 85 85 8
Absidia glauca 1021066469
100100100100100
100100100100100
Mixia osmundae 953452159
Phanerochaete carnosa 599389094
Phanerochaete carnosa 599389098
100100100100100100100100100100
Xanthophyllomyces dendrorhous 820654791
100100100100100
Taphrina deformans 501754000
Geotrichum candidum 936526884
Tuber melanosporum 296424896
9 89 89 89 89 8
Arthrobotrys oligospora 748502452
9 99 99 99 99 9
Beauveria bassiana 667657843
Aspergillus niger 966767807
Xylona heveae 1018272766
5 35 35 35 35 3
Sclerotinia borealis 563299298
4 04 04 04 04 0
Cenococcum geophilum 1046792482
100100100100100
100100100100100
9 89 89 89 89 8
100100100100100
100100100100100
Lichtheimia corymbifera 661185607
Choanephora cucurbitarum 1043302434
Parasitella parasitica 758362759
Mucor ambiguus 758351153
100100100100100100100100100100
8 88 88 88 88 8
Absidia glauca 1021064660
100100100100100
Phycomyces blakesleeanus 1026583441
Choanephora cucurbitarum 1043299401
Parasitella parasitica 758359276
Mucor ambiguus 758356794
100100100100100100100100100100
9 69 69 69 69 6
Phycomyces blakesleeanus 1026591972
6 26 26 26 26 2
Parasitella parasitica 758358030
Mucor ambiguus 758349996
100100100100100
7 97 97 97 97 9
Absidia glauca 1021055542
9 49 49 49 49 4
Absidia glauca 1021055799
100100100100100
100100100100100
Choanephora cucurbitarum 1043300048
Parasitella parasitica 758369830
Mucor ambiguus 758347160
100100100100100100100100100100
Absidia glauca 1021067301
Absidia glauca 1021057439
9 59 59 59 59 5
Rhizopus delemar 384496914
Parasitella parasitica 758369964
Mucor ambiguus 758355288
1001001001001009 99 99 99 99 9
9 49 49 49 49 4
Choanephora cucurbitarum 1043303829
Parasitella parasitica 758366043
Mucor ambiguus 758350098
100100100100100100100100100100
9 59 59 59 59 5
Lichtheimia corymbifera 661184077
Lichtheimia corymbifera 661184007
100100100100100
Phycomyces blakesleeanus 1026584315
9 79 79 79 79 7
100100100100100
100100100100100
100100100100100
Mortierella verticillata 672823448
Mortierella verticillata 672819545
100100100100100
Choanephora cucurbitarum 1043304347
Parasitella parasitica 758370387
Mucor ambiguus 758347553
100100100100100100100100100100
Absidia glauca 1021064007
Absidia glauca 1021063680
100100100100100
Phycomyces blakesleeanus 1026596624
9 89 89 89 89 8
9 79 79 79 79 7
Lichtheimia corymbifera 661175735
100100100100100
8 78 78 78 78 7
9 09 09 09 09 0
9 89 89 89 89 8
Gonapodya prolifera 1001618781
Gonapodya prolifera 1001609369
Gonapodya prolifera 1001607778
100100100100100100100100100100
Spizellomyces punctatus 1026983364
9 39 39 39 39 3
9 99 99 99 99 9
Ciona intestinalis 699241007
Branchiostoma floridae 260801711
Homo sapiens 269846998
100100100100100100100100100100
Branchiostoma floridae 260800730
Drosophila melanogaster 24653694
Diuraphis noxia 985400732
Wasmannia auropunctata 780628690
8 88 88 88 88 8
Anopheles gambiae 347969461
5 15 15 15 15 1
100100100100100
9 99 99 99 99 9
9 79 79 79 79 7
Drosophila melanogaster 7542567
Anopheles gambiae 158300972
Bombyx mori 827550902
Wasmannia auropunctata 780638008
9 99 99 99 99 97 37 37 37 37 3
100100100100100
Wasmannia auropunctata 780657087
Bombyx mori 512922456
100100100100100
Anopheles gambiae 347967067
Diuraphis noxia 985414243
9 89 89 89 89 8
Bombyx mori 512931802
Drosophila melanogaster 24580953
Anopheles gambiae 158299082
100100100100100100100100100100
100100100100100
9 89 89 89 89 8
100100100100100
Ciona intestinalis 699240982
Ciona intestinalis 699240289
9 89 89 89 89 8
Branchiostoma floridae 260815219
Branchiostoma floridae 260805642
1001001001001009 59 59 59 59 5
Diuraphis noxia 985418216
Diuraphis noxia 985403413
9 79 79 79 79 7
Diuraphis noxia 985403431
9 89 89 89 89 8
Diuraphis noxia 985414041
Diuraphis noxia 985414036
100100100100100
9 89 89 89 89 8
Wasmannia auropunctata 780636768
Bombyx mori 827554833
9 99 99 99 99 9
Drosophila melanogaster 27819917
Anopheles gambiae 158292728
100100100100100100100100100100
Diuraphis noxia 985414070
Diuraphis noxia 985414038
100100100100100
Diuraphis noxia 985414053
9 39 39 39 39 3
Diuraphis noxia 985414057
Diuraphis noxia 985414065
100100100100100
100100100100100
8 18 18 18 18 1
9 79 79 79 79 7
Bombyx mori 827544675
Bombyx mori 827550124
100100100100100
Drosophila melanogaster 45550435
7 17 17 17 17 1
Pogonomyrmex barbatus 769865637
Wasmannia auropunctata 780711803
100100100100100
Wasmannia auropunctata 780644283
Wasmannia auropunctata 780654631
Wasmannia auropunctata 780644656
100100100100100100100100100100
100100100100100
100100100100100
100100100100100
Branchiostoma floridae 260822090
8 98 98 98 98 9
100100100100100
9 29 29 29 29 2
100100100100100
Monosiga brevicollis 167533636
Chlorella variabilis 552845248
Chlorella variabilis 552816536
9 29 29 29 29 2
Chlorella variabilis 552836356
9 99 99 99 99 9
Chlorella variabilis 552827837
100100100100100
7 87 87 87 87 8
Thalassiosira pseudonana 223994297
Phaeodactylum tricornutum 219129048
100100100100100
Monosiga brevicollis 167536477
9 79 79 79 79 7
9 79 79 79 79 7
Saprolegnia diclina 669136760
Albugo laibachii 325187969
Phytophthora infestans 301097125
Phytophthora sojae 695449355
100100100100100
Phytophthora sojae 695402341
Phytophthora infestans 301097617
Phytophthora sojae 695402334
1001001001001009 89 89 89 89 8
Phytophthora sojae 695402337
100100100100100
9 99 99 99 99 9
Plasmopara halstedii 953488399
Phytophthora infestans 301097561
100100100100100
Phytophthora sojae 695402498
100100100100100
8 98 98 98 98 9
Pythium ultimum PYU1 T009339
8 88 88 88 88 8
100100100100100
Saprolegnia diclina 669140456
Saprolegnia diclina 669140450
Saprolegnia diclina 669140454
5 95 95 95 95 9100100100100100
Saprolegnia diclina 669140460
100100100100100
Aphanomyces invadens 673038668
Aphanomyces invadens 673038670
9 99 99 99 99 9
Aphanomyces astaci 698793116
100100100100100
9 99 99 99 99 9
100100100100100
6 96 96 96 96 9
Phytophthora infestans 301113774
Phytophthora sojae 695448345
100100100100100
Phytophthora sojae 695448353
100100100100100
Phytophthora sojae 695373452
Phytophthora infestans 301091169
Phytophthora infestans 301116822
100100100100100100100100100100
Pythium ultimum PYU1 T000873
Phytophthora sojae 695373456
Phytophthora infestans 301116826
1001001001001009 79 79 79 79 7
100100100100100
100100100100100
100100100100100
8 38 38 38 38 3
8 68 68 68 68 6
Vitrella brassicaformis 873219939
5 95 95 95 95 9
Thalassiosira pseudonana 223998602
Phaeodactylum tricornutum 219120020
100100100100100
Vitrella brassicaformis 873223160
7 27 27 27 27 2
Aureococcus anophagefferens 676382117
8 98 98 98 98 9
Thecamonas trahens 923135379
6 46 46 46 46 4
Fonticula alba 694544117
5 35 35 35 35 3
6 46 46 46 46 4
Trypanosoma cruzi 407834755
Trypanosoma brucei 261333342
100100100100100
8 98 98 98 98 9
Gonapodya prolifera 1001618805
Rozella allomycis 528891384
Spizellomyces punctatus 1027003031
9 79 79 79 79 79 39 39 39 39 3
Spizellomyces punctatus 1026997757
Gonapodya prolifera 1001612267
9 09 09 09 09 0
8 08 08 08 08 0
Phycomyces blakesleeanus 1026586226
Mucor ambiguus 758352025
Parasitella parasitica 758361499
9 99 99 99 99 94 84 84 84 84 8
Choanephora cucurbitarum 1043305720
9 49 49 49 49 4
Absidia glauca 1021067652
100100100100100
Lichtheimia corymbifera 661186508
Absidia glauca 1021064963
Rhizopus delemar 384497111
Choanephora cucurbitarum 1043300786
1001001001001009 69 69 69 69 6
100100100100100
Mucor ambiguus 758350881
Parasitella parasitica 758369762
100100100100100
Absidia glauca 1021062179
100100100100100
Phycomyces blakesleeanus 1026591662
100100100100100
Choanephora cucurbitarum 1043300017
Parasitella parasitica 758367096
Mucor ambiguus 758352630
9 69 69 69 69 6100100100100100
100100100100100
100100100100100
Rhizophagus irregularis 552925556
Absidia glauca 1021064557
Lichtheimia corymbifera 661176202
5 05 05 05 05 0
Mucor ambiguus 758350158
Parasitella parasitica 758367367
1001001001001005 05 05 05 05 0
Choanephora cucurbitarum 1043305522
100100100100100
100100100100100
100100100100100
100100100100100
7 67 67 67 67 6
Dictyostelium fasciculatum 470245104
Dictyostelium fasciculatum 470237160
9 69 69 69 69 6
8 98 98 98 98 9
9 59 59 59 59 5
Anncaliia algerae 630920032
Anncaliia algerae 630921415
100100100100100
Antonospora locustae ALOC 0053
100100100100100
Nematocida parisii 387593298
Nematocida sp. 378754338
100100100100100
Edhazardia aedis 402468771
8 68 68 68 68 6
8 58 58 58 58 5
Trachipleistophora hominis THOM 1192
Vavraia culicis 667634383
100100100100100
Trachipleistophora hominis THOM 3170
Vavraia culicis 667635159
100100100100100100100100100100
Pseudoloma neurophila 948277692
Trachipleistophora hominis THOM 1681
Vavraia culicis 667631643
1001001001001006 86 86 86 86 8
Pseudoloma neurophila 948278407
8 18 18 18 18 1
Pseudoloma neurophila 948277560
9 39 39 39 39 3
Pseudoloma neurophila 948277696
100100100100100
100100100100100
Spraguea lophii 523781309
Spraguea lophii 523778440
9 39 39 39 39 3
9 19 19 19 19 1
6 86 86 86 86 8
Spraguea lophii 523777633
Spraguea lophii 523777636
100100100100100
100100100100100
Nematocida parisii 387593347
Nematocida sp. 378754560
100100100100100
9 89 89 89 89 8
Rozella allomycis 528893372
Rozella allomycis 528893373
100100100100100
Rozella allomycis 528895896
Rozella allomycis 528890810
100100100100100100100100100100
Rozella allomycis 528890951
Mitosporidium daphniae 692169676
Mitosporidium daphniae 692168879
Mitosporidium daphniae 692169241
100100100100100100100100100100
Anncaliia algerae 630919083
Enterocytozoon bieneusi 269861706
Vittaforma corneae 667642354
8 28 28 28 28 2
Enterocytozoon bieneusi 269860227
Vittaforma corneae 667640528
9 99 99 99 99 9
Nosema bombycis 484856943
Nosema ceranae 300706823
Nosema apis 530542325
100100100100100100100100100100
Ordospora colligata 734518627
Encephalitozoon cuniculi 19074987
Encephalitozoon intestinalis 303391613
Encephalitozoon hellem 401828276
Encephalitozoon romalae 685882449
1001001001001009 59 59 59 59 5
9 69 69 69 69 6100100100100100
8 38 38 38 38 3
3 63 63 63 63 6
Ordospora colligata 734518628
Encephalitozoon cuniculi 19074988
Encephalitozoon intestinalis 303391615
9 79 79 79 79 7
Encephalitozoon hellem 401828278
9 79 79 79 79 7
Encephalitozoon romalae 685882451
100100100100100
100100100100100
3 93 93 93 93 9
5 45 45 45 45 4
Edhazardia aedis 402467058
Pseudoloma neurophila 948277954
Trachipleistophora hominis THOM 0963
Vavraia culicis 667635407
100100100100100100100100100100
7 77 77 77 77 7
Edhazardia aedis 402467117
9 49 49 49 49 4
4 64 64 64 64 6
Anncaliia algerae 630921337
Antonospora locustae ALOC 0069
100100100100100
6 26 26 26 26 2
Spraguea lophii 523781240
6 86 86 86 86 8
Edhazardia aedis 402467119
8 48 48 48 48 4
Nematocida parisii 387594739
Nematocida sp. 378756386
100100100100100
5 25 25 25 25 2
100100100100100
9 99 99 99 99 9
8 18 18 18 18 1
8 88 88 88 88 8
100100100100100
100100100100100
Caulobacter vibrioides 499221961
Escherichia coli NupG
Escherichia coli XapB
Beauveria bassiana 701777725
9 99 99 99 99 9100100100100100
Pseudomonas aeruginosa 872679995
Beauveria bassiana 701780680
Escherichia coli YegT
Trichuris trichiura 669216027
100100100100100100100100100100
100100100100100
9 99 99 99 99 9
100100100100100
Halanaerobium hydrogeniformans 503171911
Mahella australiensis 503545740
100100100100100
9 49 49 49 49 4
Pseudomonas aeruginosa 685897540
Ktedonobacter racemifer 495178928
9 69 69 69 69 6
Treponema primitia 497943222
9 59 59 59 59 5
Halosarcina palladia 495660784
Halorhabdus tiamatea 495801279
9 99 99 99 99 9
Halorhabdus tiamatea 495802559
100100100100100
9 59 59 59 59 5
9 99 99 99 99 9
9 99 99 99 99 9
Beauveria bassiana 701780506
100100100100100
100100100100100
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 5 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 1—figure supplement 2. Detailed phylogeny of MFS proteins shown in Figure 1—figure supplement 1. The Figure illustrates the ultrafast
bootstrap support values (1000 replicates) for all branches, full species names and the GenBank GI numbers for each protein sequence are included in
the alignment.
DOI: https://doi.org/10.7554/eLife.47037.005
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 6 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
!"#"
!"#$%&'()*+,
-.#/'+0&&1&'/,
23 8,9,25:;<=>?@
Figure 1—figure supplement 3. Comparisons of indels in the extracellular loop between the 7th and 8th transmembrane domains present in
Microsporidia MFS proteins from clade A. (A) Illustration of the ThMFS1 - THOM_0963 indel in the context of the predicted topology of the entire
protein illustrated in Figure 1—figure supplement 4. The two arrows indicate the residues at either end of the indel, see alignment in panel B. (B)
Amino acid alignment illustrating the position of the indels in Microsporidia MFS proteins of clade A (boxed in red). All investigated Microsporidia
species encode at least one protein from clade A. The indel was acquired in a common ancestor of the core Microsporidia (as defined in Corsaro et
al., 2016; Galindo et al., 2018) and was retained in most species and sequences, suggesting a possible functional role. For one protein from Anncallia
algerae the indel has been dramatically reduced in size (Locus tag H312_02678, highlighted with black arrow head).
DOI: https://doi.org/10.7554/eLife.47037.006
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Research article Evolutionary Biology Microbiology and Infectious Disease
2
3
4
EXTRACELLULAR
CYTOPLASMIC
EXTRACELLULAR
EXTRACELLULAR
EXTRACELLULAR
CYTOPLASMIC
CYTOPLASMIC
CYTOPLASMIC
Figure 1—figure supplement 4. Predicted topology of the ThMFS1-4 proteins. A topology model of the ThMFS1-4 membrane proteins is illustrated
together with its prediction statistics by the indicated program. The ThMFS1-THOM_0963, ThMFS3-THOM_1681, and ThMFS4-THOM_3170 models are
Figure 1—figure supplement 4 continued on next page
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 8 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 1—figure supplement 4 continued
based on the TOPCONS consensus sequence (Tsirigos et al., 2015). For ThMFS2-THOM_1192, TOPCONS predicted a 11 TMD topology, whereas
Philius and PolyPhobius both predict 12 TMDs and this topology is also supported by additional programs (TMHMM and HMMTOP, data not shown).
Furthermore, the close evolutionary relationship of the four proteins supports a similar structure. Therefore, we also propose a 12 TMD topology for
ThMFS2-THOM_1192 and its illustration is based on the Philius prediction. Models were visualised with TMRPres2D (Spyropoulos et al., 2004).
DOI: https://doi.org/10.7554/eLife.47037.007
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Research article Evolutionary Biology Microbiology and Infectious Disease
A B
C
3
14
22
40
70
96
ThMFS4 (THOM_3170)
ThMFS3 (THOM_1681)
ThMFS2 (THOM_1192)
ThMFS1 (THOM_0963)
−2 −1 0 1 2
Row Z−Score
Color Key
Time (hours)0
100
200
300
400
33 1414 2222 4040 7070 969633 1414 2222 4040 7070 969633 1414 2222 4040 7070 969633 1414 2222 4040 7070 9696
Time (hours)
Exp
ress
ion (TPM
)
ThMFS1 (THOM_0963)ThMFS2 (THOM_1192)ThMFS3 (THOM_1681)ThMFS4 (THOM_3170)
ThMFS1
ThMFS3
3h 14h 22h 40h 70h 96h
Figure 2. Transcription and subcellular localization of T. hominis MFS- proteins in a synchronised infection. (A) RNA-Seq data for the four ThMFS1-4
encoding genes for six time points post infection corresponding to key stages of the T. hominis infection cycle. The Y-axis shows transcripts per million
reads (TPM - Black lines indicate the value from each replicate) against time on the X-axis (hours). (B) Relative abundance of transcripts (Z-score -
normalised values based on the average TPM for each gene) for each ThMFS gene across the time points illustrated in panel A. (C) IFA data for
ThMFS1 (THOM_0963) (rabbit 94, antisera dilution 1:50, red) and ThMFS3 (THOM_1681) (rabbit 91, antisera dilution 1:50, red) proteins showing
localization to the periphery of parasites. Time points were chosen based on the following stage-specific features appearing post infection: 3 hr
germinated sporoplasm (smallest vegetative cells of the parasite, see inset for zoom in on the parasite), 14 hr unicellular meronts (see inset for zoom in
on the parasite), 22 hr first nuclear division, 40 hr first cellular division, 70 hr initiation of cellular differentiation into sporonts and spores, 96 hr fully
mature spores within the SPOV (Dean et al., 2018). Small arrows indicate labelled parasites, large arrows indicate labelled SPOV, small arrow heads
indicate unlabelled parasites (3 hr and 70 hr) or unlabelled SPOV (96 hr). Infection of new host cells from mature spores can be observed in the later
time points (an example is illustrated in Figure 2—figure supplement 9). ThMFS1 was not detectable at the first time point whereas the sporoplasms
were labelled with the ThmitHsp70 mitosomal marker (rat antisera dilution 1:200, green). Quantification of the different IFA signals (ThMFS1, ThMFS3
and mitHsp70) (Figure 2—source data 3, Figure 2—figure supplement 2) is consistent with the pattern observed in panel 2C. The nuclei of the
mammalian host cells (large nuclei) and parasites (small nuclei) were labelled with DAPI (blue). The scale bar is 2 mm.
DOI: https://doi.org/10.7554/eLife.47037.010
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 10 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
!
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03)A?,;?A3C36-B+@?A*,)*59-,3456+
D6AE39A6AF-@539G
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D6AE39A6AF-@539G
03)A?,;?A3C36-B+@?A*,)*59-,3456+
D6AE39A6AF-@539G
Figure 2—figure supplement 1. Published RNA-Seq data from the Microsporidia Vavraia culicis, Encephalitozoon cuniculi, Nematocida parisii and
Edhazardia aedis. Expression values for MFS homologues were extracted from published data for four Microsporidia species (Cuomo et al., 2012;
Figure 2—figure supplement 1 continued on next page
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Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 2—figure supplement 1 continued
Desjardins et al., 2015; Grisdale et al., 2013). Left panel: Expression (FPKM values) over time. Clade A candidates are shown in blue, clade B
candidates in red or orange. Right panel: Heat maps illustrating changes in relative expression levels (z-scores) between the investigated timepoints.
The following sequences shared between T. hominis and V. culicis are orthologous: ThMFS1-THOM_0963–VCUG_01971; ThMFS2-THOM_1192–
VCUG_01455; ThMFS3-THOM_1681–VCUG_00084; ThMFS4-THOM_3170–VCUG_01849 (see Figure 1 for phylogeny supporting orthology).
DOI: https://doi.org/10.7554/eLife.47037.011
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Research article Evolutionary Biology Microbiology and Infectious Disease
0
500
1000
1500
168h 240h
Time
Ex
pre
ssio
n (
FP
KM
)
VCUG_01971VCUG_00084VCUG_01849VCUG_01455
Spores
Color Key
250
500
750
1000
24h 48h 72h
Time
Ex
pre
ssio
n (
FP
KM
)
ECU11_1870ECU11_1880
Color Key
0
200
400
600
8h 16h 30h 40h 64h
Time
Ex
pre
ssio
n (
FP
KM
)
NEPG_00699 (≈NEQG_00533)NEPG_00147 (≈NEQG_01061)NEPG_01183 (≈NEQG_01766)
Color Key
0
300
600
900
1200
72h 120h 144h 272h 320h
Time
Ex
pre
ssio
n (
FP
KM
)
EDEG_00277EDEG_00280EDEG_00275EDEG_01832
Spores
Color Key
A
B
C
D
168h
240h
24h
48h
72h
8h
16h
30h
40h
64h
72h
120h
144h
272h
320h
Spores
Spores
Figure 2—figure supplement 2. Comparison of IFA signal quantification for ThMFS1, ThMFS3 and mitHsp70. Quantification of parasite specific IFA
signals (y-axis, arbitrary units) for ThMFS1 performed on 3–4 parasites (A), ThMFS3 performed on 3–4 parasites (B) and mitHs70 (C) performed on 5–6
Figure 2—figure supplement 2 continued on next page
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Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 2—figure supplement 2 continued
parasites for the six different time points post infection (x-axis, hours). The data for mitHsp70 combines cells (5–6 parasites) from the ThMFS1 and
ThMFS3 IFA data.
DOI: https://doi.org/10.7554/eLife.47037.012
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Research article Evolutionary Biology Microbiology and Infectious Disease
B
Th 1 - THOM_0963 (rabbit #94 and #95) Peptide #7, 17-mer CIKSYDRAERSNADIES Peptide #8, 15-mer CEDEGDNKPSNPKST
Th 2 - THOM_1192 (rabbit #88 and #89) Peptide #1, 19-mer CKTPKFKKDVKENLTREGR Peptide #2, 17-mer CIDRDLKDPRTVNEDES
Th 3 - THOM_1681 (rabbit #90 and #91) Peptide #3, 16-mer CNYLEHEGLDVRQSGR Peptide #4, 16-mer CFSRRLRGEGTKNREN
Th 4 - THOM_3170 (rabbit #92 and #93) Peptide #5, 16-mer CVKRTNSSNRNVGTAK Peptide #6, 16-mer CKPEAVLFKRKISLKD
Figure 2—figure supplement 3. Peptide designed to generate specific antisera for the ThMFS1-4 proteins. (A)
Alignment of ThMFS1-4 proteins highlighting the selected peptide sequences used to generate antisera. Two
peptides per protein were selected to optimise their solubility and specificity for a given protein. All peptides
correspond to the most soluble segments of the proteins with corresponding TMD highlighted in grey shading as
inferred by the analyses described in Figure 2—figure supplement 4. The locus tags in bold correspond to the
two proteins detected by IFA in T. hominis infected RK13 cells with the anti-peptide antisera (Figure 2C). (B)
Details of the sequence features of peptides used to generate the rabbit antisera for the four ThMFS1-4 proteins.
Red: positively charged residues. Blue: negatively charged residues. Green: hydrophobic residues. Black:
uncharged residues.
DOI: https://doi.org/10.7554/eLife.47037.013
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 15 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
A BThMFS2 ThMFS4
Ra
bb
it 8
8R
ab
bit
89
Ra
bb
it 9
2R
ab
bit
93
C
Figure 2—figure supplement 4. Comparison of the IFA signals for antisera for ThMFS1-4 and mitHsp70. Broad fields IFA images from T. hominis-
infected RK13 cells incubated with rabbit antisera (affinity purified antibodies) that did not generate parasite-specific signal: (A) ThMFS2 rabbit 88 and
89 and (B) ThMFS4 rabbit 92 and 93. A mix of early and late meronts stages (labelled with rat antisera for mitHsp70) can be observed in panels A and B.
Figure 2—figure supplement 4 continued on next page
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 16 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 2—figure supplement 4 continued
Spores can be seen within SPOVs. In panels A and B there is no parasite associated ThMFS2/4 signal (red channel) and only weak labelling of host cell-
associated structures. Panel C compares the quantification of parasite specific IFA signal (y-axis, arbitrary units) for ThMFS1-4 (red) and mitHs70 (green)
for the indicated rabbit antisera. The antisera that generated parasite-specific signals (Figure 2C) have significantly higher values for the rabbit antisera
(red channel – anti ThMFS1/3). In contrast, the signal from the rat antisera (green channel – anti mitHsp70) is more homogenous across all IFA analyses,
as expected when meront stages of the parasites are present.
DOI: https://doi.org/10.7554/eLife.47037.014
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 17 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
! ! ! ! ! ! ! !
" " " " " " " "
# # # # # # # #
$$ $% %& %' %( %) %* %+,-../01
2&
2&++
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)+)+
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3456#) 3456#'73456#( 3456#*
Figure 2—figure supplement 5. Western blot analysis on total protein extracts from host cells and parasites with antisera against ThMFS1-4. Affinity
purified antibodies (from rabbit antisera 88–95, 1/1000 dilution) were used for Western blot analyses on total protein extracts from RK13 cells (H, 10 mg
proteins), T. hominis infected RK13 cells (I, 10 mg proteins) and purified T. hominis spores (S, 5 mg proteins). The two sets of antisera that give parasite
specific IFA signals are indicated in red. Conspicuous parasite specific bands are indicated by arrow heads. Black arrowheads highlight bands with
apparent Mw that correspond approximately to full length ThMFS proteins (calculated Mw: ThMFS1, 55 kDa and ThMFS3, 50 kDa). Blue arrowheads
highlight potential degradation products of the full-length proteins. Some bands are not parasite specific as they appear in both control non-infected
cells (H) and parasite-infected cells (I). The antisera for ThMFS2 (Rabbit 88 and 89) and ThMFS4 (rabbit 92 and 93) that gave no parasite-specific IFA
signal are also characterised by the absence of parasite-specific signals in the western blot. The Mw (kDa) of the pre-stained markers are indicated on
the left.
DOI: https://doi.org/10.7554/eLife.47037.015
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 18 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
!" '()(!$*+,--.'(
/0(11(
/0(1.(
/0(.2(
/0(.-(
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/0(.7(
Figure 2—figure supplement 6. Dot blots on peptides to test the specificity of anti-ThMFS1-4 rabbit antibodies. Affinity purified antibodies from
rabbit antisera were diluted 1:1000 for Western blot analyses on nitrocellulose membranes. For each pair of peptides two rabbits were immunised with
the numbers Rb 88–95 specifying individual rabbits. 300 ng of each peptide was deposited on the membrane and then processed for immunodetection
with the specified rabbit antisera (Rb 88–95) in combination with goat anti-rabbit secondary antibody conjugated to HRP (dilution 1:10000).
DOI: https://doi.org/10.7554/eLife.47037.016
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 19 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 2—figure supplement 7. ThMFS3 IFA detection in highly infected RK13 cells containing mixed stages of the parasite infection cycle. The affinity
purified antibodies for ThMFS3 were used to label highly infected RK13 cells following several days of infection. A mix of cellular stages can be seen
including early sporoplasms and meronts (small arrows), later meront with weak signal (equivalent to 40 hr in Figure 2C, one cell, large arrow), and later
meront stage with no signal (larger cell equivalent to 70 hr in Figure 2C, one cell, large arrow head) differentiating sporonts in SPOV (with DAPI
labelled nuclei, stars) and mature spores in SPOV (with no labelled nuclei, stars). These data further support the variation of ThMFS3 IFA signal
observed across the time points illustrated in Figure 2C., including the loss of the signal for ThMFS3 in the last two time points (70 hr and 96 hr post
infection).
DOI: https://doi.org/10.7554/eLife.47037.017
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 20 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease
96h
Figure 2—figure supplement 8. Evidence for re-
infections from germination of newly formed mature
spores in the late time point post infection (96 hr) from
IFA for ThMFS1 and ThMFS3. Panels are distinct fields
from the same sample shown in Figure 2C illustrating
the appearance of additional cellular stages of the
parasite in the 96 hr post infection time point indicating
re-infection from newly mature spores that underwent
germination. Both the ThMFS1 and ThMFS3 antisera
label the early stage meronts present at 96 hr post
infection. For ThMSF1, the size of labelled cells is
reminiscent of those observed at 14 hr post-infection
(larger cells compared to those observed 3 hr post-
infection), whereas for ThMFS3 the cell size is
reminiscent of those observed at 3 hr post-infection
(smaller cells compared to those observed 14 hr post-
infection). Scale bar 2 mm.
DOI: https://doi.org/10.7554/eLife.47037.018
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Research article Evolutionary Biology Microbiology and Infectious Disease
- peptides + peptides
Figure 2—figure supplement 9. Peptide competition experiments demonstrate the specificity of antisera against
the ThMFS1 and ThMFS3 proteins. Rabbit antisera were used at a 1:50 dilution to detect the ThMFS1 and ThMFS3
proteins respectively. Addition of peptides that were used as antigens for antibody production (see Figure 2—
figure supplement 3 for details) at a 200x molar excess resulted in the complete loss of IFA signal for the ThMFS
(red) but not mitHsp70 (green), supporting the specificity of the respective affinity purified anti ThMFS antibodies.
DOI: https://doi.org/10.7554/eLife.47037.019
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Research article Evolutionary Biology Microbiology and Infectious Disease
ATP GTP CTP UTP
*
*
*
* *
*
*
*
ns ns
*
ns
A B
C
ptrc99a
NupG
ThMFS1
ThMFS2
ThMFS3
ThMFS4
0
10
20
30
40
50
[32P]ATPpmol.mg-1.(30min)-1
2 µM ATP30 minutes
Figure 3. Transport assay for the nucleoside uridine and selected nucleotides in E. coli expressing recombinant E. coli NupG, ThMFS1-4 proteins or
Rozella allomycis NTT. (A) Radiolabelled uridine uptake assay with E. coli cells expressing the native E. coli NupG transporter or one of the four ThMFS
Figure 3 continued on next page
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Research article Evolutionary Biology Microbiology and Infectious Disease
Figure 3 continued
proteins (ThMFS1-4) cloned into the expression vector ptrc99a. The empty ptrc99a plasmid was used as a control for background transport of the
radiolabelled substrate. (B) Radiolabelled ATP import assay for the same five genes as in (A) and with the same control of the empty ptrc99a plasmid.
(C) Uptake assays for the four radiolabelled nucleotides (a32P-) ATP, GTP, CTP and UTP using the same expression system as in A and B. The same
control (empty ptrc99a plasmid) was used for each tested substrate. For the Rozella NTT (RozNTT) cloned in pET16b, the control was the empty
plasmid pET16b (Dean et al., 2018). N = 3 for each condition and the error bars represent standard deviations. Significant differences at p<0.05 (one-
way ANOVA) between controls (empty plasmids, ptrc99a or pET-16b) and individual transporters are shown with * (ns: non-significant).
DOI: https://doi.org/10.7554/eLife.47037.023
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Research article Evolutionary Biology Microbiology and Infectious Disease
A
BThMFS2
ptrc99a ATP
ThMFS2 ATP
ptrc99a GTP
ThMFS2 GTP
ThMFS3
pET-16b ATP
ThMFS3 ATP
pET-16b GTP
ThMFS3 GTP
ThMFS1
pET-16b ATP
ThMFS1 ATP
pET-16b GTP
ThMFS1 GTP
ThMFS4
ptrc99a ATP
ThMFS4 ATP
ptrc99a GTP
ThMFS4 GTP
Figure 4. Time course of ATP and GTP uptake by E. coli cells expressing recombinant ThMFS1-4 proteins. Each ThMFS transporter was assayed using
both E. coli-expression vector systems (pET16b or ptrc99a) with the results shown being taken from the experiment with the highest transport activity.
In each experiment, the corresponding empty plasmid was used as control for background transport. The indicated substrates were all used at 0.5 mM.
(A) Uptake assay for the ThMFS1 (THOM_0963) protein expressed using pET16b in E. coli Rosetta2(DE3)pLysS and the native (not codon optimised)
ORF. (B) Uptake assay for the ThMFS2 (THOM_1192) protein expressed with the ptrc99a plasmid system in E. coli GD1333 and the E. coli codon
optimised synthetic ORF. (C) Uptake assay for the ThMFS3 (THOM_1681) protein expressed with pET16b system in E. coli Rosetta2(DE3)pLysS and the
native ORF. (D) Uptake assay for the ThMFS4 (THOM_3170) protein expressed with the ptrc99a plasmid system in E. coli GD1333 and the E. coli codon
optimised synthetic ORF. N = 3 for each condition with the error bars representing standard deviations. All 8 min time points for specified transporters
and nucleotides were significantly different at p<0.05 (one-way ANOVA) from controls (empty plasmids, ptrc99a or pET-16b).
DOI: https://doi.org/10.7554/eLife.47037.025
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Research article Evolutionary Biology Microbiology and Infectious Disease
ThMFS1ATP
ThMFS3ATP
ThMFS1GTP
ThMFS3GTP
PamNTT5GTP
0
50
100
150
%ofcontrol
0.5 µM NTP+CCCP10 minute assay
*
Figure 5. Lack of impact of the protonophore CCCP on nucleotide import in E. coli expressing the two parasite
cell-surface located ThMFS proteins. [a32P]-nucleotide import by ThMFS1, ThMFS3, and by the Protochlamydia
amoebophila symporter NTT5 (PamNTT5) (Haferkamp et al., 2006) was compared in the absence (control, set to
100%) and presence of the protonophore CCCP. The GTP and ATP H+-symporter PamNTT5 was used as a
positive control for CCCP inhibition (Haferkamp et al., 2006). N = 3 for each condition with the error bars
representing standard deviations. The significant reduction of transport between control (no CCCP) and CCCP
treatments at p<0.05 (one-way ANOVA) is indicated with *.
DOI: https://doi.org/10.7554/eLife.47037.027
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Research article Evolutionary Biology Microbiology and Infectious Disease
pET-16b
ThMFS1
ThMFS3
pET-16b
ThMFS1
ThMFS3
pET-16b
ThMFS1
ThMFS3
PamNTT5
pET-16b
ThMFS1
ThMFS3
PamNTT5
0
20
40
60
[32P]NTPpmol.mg-1.(10min)-1
ATP ATP+CCCP GTP GTP+CCCP
0.5 µM NTP10 minutes
ns
*
*
*
*
*
*
*
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*
*
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Figure 5—figure supplement 1. Impact of the protonophore CCCP on nucleotide transport by E. coli expressing ThMFS1, ThMFS3 or the control
PamNTT5. The transport data used to generate Figure 5. Uptake assays were performed using radiolabelled nucleotides in presence or absence of the
protonophore CCCP. The drug did not inhibit nucleotide uptake in E. coli cells expressing ThMFS1 or ThMFS3. Inhibition of GTP uptake by PamNTT5
expressing E. coli cells (positive control; Haferkamp et al., 2006) demonstrated that the drug was functional. Significant differences at p<0.05 (one-way
ANOVA) between controls (empty plasmid) and indicated transporters are shown with blue stars (*) or between untreated and CCCP treatments are
shown with black stars (*) (the black line highlights the comparison for the PamNTT5 transporter). N = 3 with error bars representing standard deviation.
DOI: https://doi.org/10.7554/eLife.47037.028
Major et al. eLife 2019;8:e47037. DOI: https://doi.org/10.7554/eLife.47037 27 of 27
Research article Evolutionary Biology Microbiology and Infectious Disease