major, p., sendra, k. m., dean, p. , williams, t., watson

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

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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


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Amphiamblys sp (A8A55_0782)Amphiamblys sp (A8A55_2217)

Nematocida parisii (NEQG_01061)Nematocida sp (NERG_02401)


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


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


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



https://doi.org/10.7554/eLife.47037.003


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.






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





100100100100100100100100100100

8 88 88 88 88 8


100100100100100





100100100100100100100100100100

9 69 69 69 69 6


6 26 26 26 26 2



100100100100100

7 97 97 97 97 9


9 49 49 49 49 4


100100100100100

100100100100100




100100100100100100100100100100



9 59 59 59 59 5




1001001001001009 99 99 99 99 9

9 49 49 49 49 4




100100100100100100100100100100

9 59 59 59 59 5



100100100100100


9 79 79 79 79 7

100100100100100

100100100100100

100100100100100

Mortierella verticillata 672823448

Mortierella verticillata 672819545

100100100100100




100100100100100100100100100100



100100100100100


9 89 89 89 89 8

9 79 79 79 79 7


100100100100100

8 78 78 78 78 7

9 09 09 09 09 0

9 89 89 89 89 8

Gonapodya prolifera 1001618781



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


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



Bombyx mori 827550902


9 99 99 99 99 97 37 37 37 37 3

100100100100100



100100100100100



9 89 89 89 89 8




100100100100100100100100100100

100100100100100

9 89 89 89 89 8

100100100100100



9 89 89 89 89 8



1001001001001009 59 59 59 59 5



9 79 79 79 79 7


9 89 89 89 89 8



100100100100100

9 89 89 89 89 8



9 99 99 99 99 9



100100100100100100100100100100



100100100100100


9 39 39 39 39 3



100100100100100

100100100100100

8 18 18 18 18 1

9 79 79 79 79 7



100100100100100


7 17 17 17 17 1

Pogonomyrmex barbatus 769865637


100100100100100




100100100100100100100100100100

100100100100100

100100100100100

100100100100100


8 98 98 98 98 9

100100100100100

9 29 29 29 29 2

100100100100100

Monosiga brevicollis 167533636

Chlorella variabilis 552845248


9 29 29 29 29 2


9 99 99 99 99 9


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




1001001001001009 89 89 89 89 8


100100100100100

9 99 99 99 99 9

Plasmopara halstedii 953488399


100100100100100


100100100100100

8 98 98 98 98 9

Pythium ultimum PYU1 T009339

8 88 88 88 88 8

100100100100100




5 95 95 95 95 9100100100100100


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



100100100100100


100100100100100




100100100100100100100100100100

Pythium ultimum PYU1 T000873



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




9 79 79 79 79 79 39 39 39 39 3



9 09 09 09 09 0

8 08 08 08 08 0




9 99 99 99 99 94 84 84 84 84 8


9 49 49 49 49 4


100100100100100





1001001001001009 69 69 69 69 6

100100100100100



100100100100100


100100100100100


100100100100100




9 69 69 69 69 6100100100100100

100100100100100

100100100100100

Rhizophagus irregularis 552925556



5 05 05 05 05 0



1001001001001005 05 05 05 05 0


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


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



100100100100100100100100100100

Pseudoloma neurophila 948277692



1001001001001006 86 86 86 86 8


8 18 18 18 18 1


9 39 39 39 39 3


100100100100100

100100100100100

Spraguea lophii 523781309


9 39 39 39 39 3

9 19 19 19 19 1

6 86 86 86 86 8



100100100100100

100100100100100



100100100100100

9 89 89 89 89 8



100100100100100



100100100100100100100100100100


Mitosporidium daphniae 692169676



100100100100100100100100100100


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





100100100100100100100100100100

7 77 77 77 77 7


9 49 49 49 49 4

4 64 64 64 64 6


Antonospora locustae ALOC 0069

100100100100100

6 26 26 26 26 2


6 86 86 86 86 8


8 48 48 48 48 4



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


9 99 99 99 99 9100100100100100

Pseudomonas aeruginosa 872679995


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


100100100100100

100100100100100




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.






!"#"

!"#$%&'()*+,

-.#/'+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).






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




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).






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.






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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;






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).






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






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.






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.






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.






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.






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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.






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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).






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).






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.






- 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.






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




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).






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).






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 *.






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

*

*

*

*

*

*

*

**

*

*

*

*

*

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.






major, p., sendra, k. m., dean, p. , williams, t., watson

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