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C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence

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C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence

ALBUQUERQUE-WENDT, Andreia, et al.

ALBUQUERQUE-WENDT, Andreia, et al . C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence. Journal of Biological Chemistry , 2020, p. jbc.RA119.010590

PMID : 31862733

DOI : 10.1074/jbc.RA119.010590

Available at:

http://archive-ouverte.unige.ch/unige:128181

Disclaimer: layout of this document may differ from the published version.

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Signal intensity 200 400 600

Signal intensity 50 100 150 200

0

1216 1217 1218 1219 1220 Signal intensity 50

100 150 200

1215

1217.57 1218.06 1217.03

1377 1378 1379 1380 1381 1382 1383

1379.63 1380.12 1379.11

mass (m/z)

1296 1297 1298 1299 1300 1301 1302

1298.56 1299.10 1298.07

mass (m/z) mass (m/z)

B

MIC2 peptide DERPGEWAEWGECSVTCG

A

DERPGEWAEWGECSVTCG [M+2H] = 1298.07; +2 Hex +1 Fuc; 25.7 min

2+

DERPGEWAEWGECSVTCG [M+2H] = 1217.03; +1 Hex +1 Fuc; 27.9 min

2+

DERPGEWAEWGECSVTCG [M+2H] = 1379.11; +3 Hex +1 Fuc; 25.1 min

2+

DERPGEWAEWGECSVTCG [M+2H] = 1135.95; +0 Hex +1 Fuc

2+

24 25 26 27 28 29

2 Signal intensity (x 10)

5 4 3 2 1 0

24 25 26 27 28 29

2 Signal intensity (x 10)

5 4 3 2 1 0

Wild type Δdpy19

Time (in min) Time (in min)

DSSGSIGIQNFR ADWGQLATAIKPMLK

MIC2 peptide

ADWGQLATAIKPMLK

C

3 Signal intensity (x 10)

0 1 2 3

3 Signal intensity (x 10)

0 1 2 3 4

mass (m/z)

3 Signal intensity (x 10)

0 1

Δdpy19

2

DSSGSIGIQNFR

mass (m/z)

640.85

641.35

641.86

640.88 641.39

641.88

638 639 640 641 642 643 644 WT

Δdpy19 Δdpy19

Time (in min)

26.99 26.88

29.75

29.75

26 27 28 29 30 31 32

WT

WT

Δdpy19

827 828 829 830 831 832 833 WT

Figure S1: Analysis of MIC2 peptides from T. gondii wild type and Δdpy19 strains. T. gondii wild type and Δdpy19 parasites were lysed and proteins separated by SDS-PAGE. The MIC2 containing bands were excised and proteins were reduced, alkylated with iodoacetamide and digested with trypsin and AspN. The resulting peptides were separated on a nano Ultra Performance Liquid Chromatography and analyzed by tandem mass spectrometry on an ESI-Q-TOF Ultima. A) Extracted Ions chromatograms of [M+2H] ions at m/z 1379.03;

2+

1298.00; 1216.98 and 1135.95 corresponding to the glycopeptide DERPGEWAEWGECSVTCG. Assignment shown by the schematic is based on peptide mass and retention time. Other glycoforms were not observed. B) Mass spectra of [M+2H] ions observed in wild type

2+

parasites. C) Extracted ion chromatograms and mass spectra of two MIC2 peptides demonstrating the presence of MIC2 in samples from wild

type (WT) and the Δdpy19 strain.

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S2 Table S1: Prediction of Toxoplasma gondii DPY19 substrate proteins

Gene information WX2W

X2C motifs

Fitness Score b

Protein information c Transcriptomic (RNA-seq): Read counts d

Gene product a Gene ID TMD MW

(kDa)

Signal peptide

Asexual Stages Sexual Stages

Tachyzoites Bradyzoites Merozoites Gametes Oocysts

Microneme protein MIC2 TGGT1_201780 6 -1.17 1 82.6 Yes 743.04 231.08 0.90 0.49 0.11

Sushi domain containing protein TGGT1_223480 15 -3.90 1 507.0 No 1.18 1.05 5.38 10.90 24.31

TSP1 domain-containing protein TGGT1_209060 6 -1.70 1 93.6 Yes 22.19 6.19 14.06 18.08 14.12

Microneme protein MIC15 TGGT1_247195 3 -3.31 1 320.0 Yes 46.46 22.43 0.65 3.15 8.26

Microneme protein MIC14 TGGT1_218310 3 1.30 1 105.9 No 75.20 67.22 58.74 53.79 43.62

Microneme protein MIC12 TGGT1_267680 2 0.50 0 233.8 Yes 16.72 104.37 69.04 321.67 99.43

Microneme protein MIC16 TGGT1_289630 2 1.98 1 74.6 Yes 59.29 43.14 0.16 0.45 0.38

Hypothetical protein TGGT1_282000 2 -0.56 2 208.8 No 26.06 13.51 1.79 12.98 50.34

TSP1 domain-containing protein TGGT1_277910 1 -2.95 1 134.4 Yes 11.60 13.01 4.32 7.36 4.06

Hypothetical protein TGGT1_319992 1 -0.48 0 24.5 No 1.60 2.22 2.12 6.71 14.48

Hypothetical protein TGGT1_247970 1 -0.94 2 135.9 Yes 0.28 0.05 0.57 5.98 12.48

Hypothetical protein TGGT1_237585 1 1.41 1 139.1 No 0.12 2.61 1.97 10.16 20.38

Hypothetical protein TGGT1_210700 1 -1.95 0 926.5 No 4.11 3.77 1.23 1.32 1.10

α-tubulin suppressor protein TGGT1_267450 1 0.77 2 104.2 No 25.35 12.14 4.42 11.36 5.17

a

Proteins with at least one WX*XWX*XW motif with X* being a serine, alanine, glycine or threonine have been selected as putative C-mannosylated proteins.

b

Fitness score as determined in (52). A red background indicates a gene essential for infection of host cells while a blue background indicates a dispensable gene.

c

TMD: number of transmembrane domain predicted with TMHMM-2.0. Molecular weight (MW) in kDa and prediction of signal peptide with SignalP-5.0 (http://www.cbs.dtu.dk/services).

d

The transcriptomics data have been retrieved from Adrian B. Hehl's (University of Zurich, Switzerland) dataset “Transcriptomes of Toxoplasma gondii strain

CZ clone H3 in feline enteroepithelial stages”. For each gene, the stage with the most expression appears with a green background and the stage with the least

expression appears with a red background.

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S3

Table S2: Sequence of WX

2

WX

2

C motifs in putative Toxoplasma gondii C-mannosylated proteins

Protein name a Encoding gene Sequence of WX2WX2C motif Microneme protein MIC2 b TGGT1_201780 WSAWSPC283

FEEWSTC482

WGEWSAWSASC355

WAEWGEC543

WAAWSDC415

WSDWNKC610

Sushi domain containing protein TGGT1_223480

WNEWSNC484 WSDWSSC652 WEDWGEC815 WTEWTAC1058 WTDWTPC1286

WGEWSDC542 WTPWSAC705 WGAWGPC876 WNDWTGC1117 WGEWSTC1350

WSEWGPC597 WSEWSVC760 WGAWSSC1003 WGEWTGC1177 WSPWSSC1398 TSP1 domain-containing protein TGGT1_209060 WSEWGAC164

WEEWSAC522

WGDWSPC348 WTTWSAC593

WSAWSPC409 WQPWTAC668 Microneme protein MIC15 TGGT1_247195 WGEWSAC1782 WTEWSEC1840 WSEWSTC2511

Microneme protein MIC14 TGGT1_218310 WTSWSKC155 WGDWSGC218 WSEWSEC643 Microneme protein MIC12 TGGT1_267680 WTEWTYC119 WGPWSPC1584

Microneme protein MIC16 TGGT1_289630 WQEWQSWSSC442 WTAWSDC584 Hypothetical protein TGGT1_282000 WSEWSSC695 WSSWSSC1032

TSP1 domain-containing protein TGGT1_277910 WSEWTSC664 Hypothetical protein TGGT1_319992 WSEWTAC118 Hypothetical protein TGGT1_247970 WSEWSLC886 Hypothetical protein TGGT1_237585 WTEWTTC944

Hypothetical protein TGGT1_210700 WARWSGC6506 α-tubulin suppressor protein TGGT1_267450 WGAWGSC901

a Proteins with at least one WX*XWX*XW motif with X* being a serine, alanine, glycine or threonine have been selected as putative C-mannosylated proteins.

b C-mannosylation of tryptophan residues marked in green has been confirmed (31,32,39)

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S4

Table S3: Primers utilized for generation and characterization of T. gondii mutants

Primer name Primer sequence (5´-3´)

2018 CTTGGGGGTCATCGCGACGACCAGAC

2017 GTCACTTGTTGTGCCAGTTCTAC

AFA01 GACTCAAAGGAGGTGGCGAA

AFA02 GAAGTTAGCCGCAAACACGG

AFA65 GTCGAAGCAGAGAAGGAACTC

AFA96 GCTTGCACCTATTCAGACTCGCTCTCGAAAGCGGCCGCTCTAGAACTAG AFA97 AGGATCTTTTTTCCCGGAGAAGGAAGATTAGCGGAAGATCCGATCTTGC

AFA102 GCTAGGTCTCCAAGTTGCAGACTCGCTCTCGAAAATAGTTTTAGAGCTAGAAATAGC AFA103 GCTAGGTCTCCAAAACAGTTAATCTTCCTTCTCCGGCAACTTGACATCCCCATTTA AFA113 CCCCATCCTGGAACCTTGCG

AFA114 GCTATTAATTAATAGGAAAGAAGGAGGGTATT AFA115 GCTAATGCATATGGCGAAAGTCCGAGTGGA AFA116 GATATCCGACGGTATCGATAAGCTTG AFA118 AGATCTGCTCTAGAACTAGTGGATCC DSF4883 AACTTGACATCCCCATTTAC

DSF8282 GACGAGGACGAGGCCAGCAAAGATGACAAGGCTAGCAAGGGCTCGGG DSF8283 CTCTCTGTTTTTGCCCCCGTTTCTGATGAGATACGACTCACTATAGGG DSF8284 GAGCCGCGTCGCAGGAAAAGCGTTTTAGAGCTAGAAATAGC

DSF8285 GCCGTGACACCCGAATCCTTTGGGTCCATGGCTAGCAAGGGCTCGGG DSF8286 CAGGGAGAAACGCGAGGCGTTCGCGTCAAAATACGACTCACTATAGGG DSF8287 AGACAAACGGTGGGGTGTAGGTTTTAGAGCTAGAAATAGC

DSF5272 GCCGAATTCGTATCAAAATGTCTGCCTCTCTCATG

DSF5273 GGCATGCATCCTTCTCCACTCTCTTCAGCTC

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