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Intragenic repeats generate size variability in immunogenic proteins in Aspergillus fumigatus

1. Intragenic repeats generate size variability in immunogenic proteins in Aspergillus fumigatus. 2. Nir Osherov Human Microbiology Department, Tel Aviv University. Satellite. Microsatellite. <150b Variable locations. 100Kb-1Mb Centromere. Non-coding. Coding. 3.

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Intragenic repeats generate size variability in immunogenic proteins in Aspergillus fumigatus

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  1. 1.

  2. Intragenic repeats generate size variability in immunogenic proteins in Aspergillusfumigatus 2. Nir Osherov Human Microbiology Department, Tel Aviv University

  3. Satellite Microsatellite <150b Variable locations 100Kb-1Mb Centromere Non-coding Coding 3. What is repetitive DNA? Minisatellite 1Kb-20Kb Telomere Three types:

  4. Example of tandem repeat containing protein 4. DNA Afu2g5150 Protein Variable protein

  5. N- -C leader GPI-anchor motif Tandem- repeats Ser/Thr-rich S.cerevisiae FLO1 adhesin 5.

  6. Adhesion increases…. Flocculation increases… Verstrepen et al., Nature Genetics 2005 6. What is the effect of tandem repeat elongation on adhesion? FLO1 is a GPI-anchored tandem-repeat protein involved in adhesion in S. cerevisiae As the number of repeats increases…

  7. 7. Does variability exist in the population? Different S. cerevisiae strains carry FLO1 alleles of varying lengths S. cerevisiae strain Verstrepen et al. Nature Genetics 2005

  8. 8 Aims of research • Are there tandem repeats in A.fumigatus genes and are they size-variable? • Are there tandem repeat-containing CWP’s in A.fumigatus and are they size-variable? • How will their deletion affect the phenotype?

  9. Tandem repeats – A. fumigatus 9. Tandem repeats ‘ETANDEM’ A. fumigatus annotated genome 292 ORFs containing Tandem Repeats (Etand>35) Kevin Verstrepen, Gerry Fink, MIT

  10. Gene name Rpt start Rptend Etandem score Rpt size Rpt # Conservation % Afu4g09600 789 1382 292 198 3 91.2 Afu2g05150 715 1104 301 39 10 93.6 Afu3g08990 613 1188 432 18 32 89.1 10. How does the data look?

  11. Aspergillus isolates Genomic DNA PCR TR-gene 1 2 3 4 5 Aspergillus isolate # 11. Are these tandem-repeat (TR) genes variable in size among A. fumigatus isolates? 1 2 3 4 5

  12. Leader Leader + GPI Non leader +GPI - Afu1g01020 Afu3g08990 + Afu7g07100 ND‡ - Afu2g18090 Afu2g05150 + Afu7g08290 + ND‡ Afu7g08500 Afu4g09600 + Afu7g08310 + - Afu3g07400 Afu6g14090 + Afu6g09340 + + Afu6g06670 Afu4g03240 - Afu4g10350 - + Afu2g03540 Afu7g00970 - Afu7g08240 - - Afu8g00630 Afu3g00270 - Afu8g06150 + + Afu5g06880 Afu1g16190 - Afu6g09360 + - Afu8g07170 Afu3g00880 - Afu5g15090 + - Afu5g11300 Afu2g07800 - Afu7g07060 + 12. Summary

  13. fumigatus GPI anchored genes • with high ETANDEM scores have • size-variable repeats 13. 1035bp Afu3g08990 Score=432 Afu2g05150 (MP-2) Score=301 1001 bp Afu4g09600 Score=292 715 bp Afu6g14090 CFEM Score=134 690bp Strain: 1 2 3 4 5 6 7 8 9 10 Af293

  14. Top-scoring A. fumigatus GPI-anchoredTR genes transcribe size-variable mRNA 14.

  15. Cons. Hypoth. CFEM Cons. Hypoth. MP-2 immunogenic secreted. Cons. Hypoth. 15. GPI-anchored TR genes encode for discrete numbers of in-frame repeats

  16. Δ Δ Δ Top scoring GPI & leader-containing genes at different time points 16.

  17. 17. Afu3g08990 in silico analysis P/T/S rich N-terminal region (~PGQPSV)18-32 six aa C-terminal rpts Extended rod domain of collagen

  18. 18. Afu3g08990-like genes are Aspergillus-specific and extremely divergent tBlastN fungal E-value gb|AAHF01000002.1| Aspergillus fumigatus 00. ref|NZ_AAKE03000010.1| Neosartorya fischeri 9e-26 ref|NZ_AAKD03000018.1| Aspergillus clavatus 6e-09 gb|AACD01000051.1| Aspergillus nidulans 3e-06 gb|AAJN01000190.1| Aspergillus terreus 8e-04 gb|AAIH01000116.1| Aspergillus flavus 8e-04 ref|NZ_AAFU01001164.1| Chaetomium globosum 0.002 ref|NC_005785.3| Ashbya gossypii 0.011 gb|AAEG01000002.1| Saccharomyces cerevisiae 0.041

  19. 19. A. fumigatus Afu3g08990 Neosartorya fischeri 9e-26 Aspergillus clavatus 6e-09 Aspergillus nidulans 3e-06 Aspergillus terreus 8e-04

  20. TR region 430 aa Afu3g08990 1562 aa Lactobacillus hyp. 10-32 1369 aa Tetrahymena FH2 10-27 1212 aa Strep. agalactiae 10-27 C-protein β-ag 940 aa Tryp. cruzi trans-sialidase 10-20 954 aa Human collagen type XXI 10-17 20. Pan-species Blast reveals little….

  21. 21. Afu3g08990 expression is upregulated by damage to the cell wall and plasma membrane

  22. Hyphal length in µm ∆1 ∆2 ∆3 random AF 293 KI Time (h) 22. Deletion of ∆Afu3g08990 leads to early germination

  23. Conidia per field AF 293 ∆1 ∆2 ∆3 random KI 23. The ∆Afu3g08990 strains exhibit reduced adhesion to ECM

  24. Afu3g08990 repeat region can be used for strain typing Balajee et al. 2007, submitted.

  25. Summary Afu3g08990 24. • Transcribed during germination and vegetative growth • Upregulated by cell-wall stress • ΔAfu3g08990 conidia germinate faster than WT • ΔAfu3g08990 conidia show reduced adherence to ECM • ΔAfu3g08990 mutant NOT more susceptible to CAS, • CR, CFW • No change in virulence (mouse tail vein model)

  26. 1. Receptor? 2. Cross-linker ‘cement’ ? PM 3. Reservoir model 25. What does Afu3g08990 do?

  27. Kobi Romano Emma Levdansky Yana Shadkchan Haim Sharon 26. I would like to thank Collaborators: Kevin Verstrepen, Harvard Gerry Fink, Whitehead Inst.

  28. 28. Thank you !

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