1.

Intragenic repeats Intragenic repeats generate size generate size variability in variability in

immunogenic proteins immunogenic proteins in in Aspergillus Aspergillus

fumigatusfumigatusNir OsherovNir Osherov

Human Microbiology Department, Tel Aviv Human Microbiology Department, Tel Aviv UniversityUniversity

2.

What is repetitive DNAWhat is repetitive DNA??

Three types:

SatelliteSatellite100Kb-1Mb

Centromere

MicrosatelliMicrosatellitete<150b

Variable locationsCoding

Non-coding

MinisatelliteMinisatellite

1Kb-20Kb1Kb-20Kb

TelomereTelomere

3.

Example of tandem repeat Example of tandem repeat containing proteincontaining protein

PTETSTPCETTTTTTETSTPCETTTTTTPPPETSTPCETTTTTTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTPPPETSTPCETTTTPTETSTPCETTTSPYRE

CCTACCGAGACCTCCACTCCGTGCGAGACTACTACCACTACTACCGAGACCTCTACCCCCTGTGAGACTACTACTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGCGAGACTACTACCACTACTACCGAGACCTCCACTCCATGTGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTACCCCTACCGAGACCTCTACTCCGTGTGAGACTACCACCACTCCTACCGAGACCTCTACTCCGTGTGAGACCACTACCACTCCTACCGAGACCTCTACTCCATGCGAGACTACCACCACTCCTACCGAAACCTCTACCCCCTGTGAGACTACTA CTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGCGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTAGCCCCTACCGAGAG

DNA

Afu2g5150

Protein

Variable protein

4.

S.cerevisiae FLO1S.cerevisiae FLO1 adhesinadhesin

leader GPI-anchor motif

N- -C

Ser/Thr-richTandem-repeats

5.

What is the effect of tandem What is the effect of tandem repeat elongation on repeat elongation on

adhesionadhesion??FLO1 is a GPI-anchored tandem-repeat protein involvedin adhesion in S. cerevisiae

Adhesion increases….

Flocculation increases…

Verstrepen et al., Nature Genetics 2005

As the number of repeats increases…

6.

Does variability exist in the Does variability exist in the populationpopulation ? ?

Different S. cerevisiae strains carry FLO1 allelesof varying lengths

S. cerevisiae strain

7.

Verstrepen et al. Nature Genetics 2005

Aims of researchAims of research Are there tandem repeats in Are there tandem repeats in A.fumigatus A.fumigatus

genes and are they size-variable?genes and are they size-variable?

Are there tandem repeat-containing Are there tandem repeat-containing CWP’s in CWP’s in A.fumigatus A.fumigatus and are they size-and are they size-variable?variable?

How will their deletion affect the How will their deletion affect the phenotype?phenotype?

8

Tandem repeats – Tandem repeats – A. A. fumigatusfumigatus

Kevin Verstrepen, Gerry Fink, MIT

292 ORFs containing Tandem Repeats(Etand>35)

Tandem repeats ‘ETANDEM’

A. fumigatus

annotated genome

9.

How does the data lookHow does the data look??

89.132184321188613Afu3g08990

93.610393011104715Afu2g05150

91.231982921382789Afu4g09600

Conservation %

Rpt #

Rpt

sizeEtandem score

Rpt

endRpt start

Gene name

10.

Are these tandem-repeat (TR) genes variable in size among A. fumigatus isolates?

Aspergillus isolates

Genomic DNA

PCR TR-gene

1 2 3 4 5

1 2 3 4 5 Aspergillus isolate #

11.

SummarySummary

Afu2g07800

Afu3g00880

Afu1g16190

Afu3g00270

Afu7g00970

Afu4g03240

Afu6g14090

Afu4g09600

Afu2g05150

Afu3g08990

-

-

-

-

-

-

+

+

+

+

Afu5g11300

Afu8g07170

Afu5g06880

Afu8g00630

Afu2g03540

Afu6g06670

Afu3g07400

Afu7g08500

Afu2g18090

Afu1g01020

-

-

+

-

+

+

-

ND‡

-

-

Afu7g07060

Afu5g15090

Afu6g09360

Afu8g06150

Afu7g08240

Afu4g10350

Afu6g09340

Afu7g08310

Afu7g08290

Afu7g07100

+

+

+

+

-

-

+

+

+

ND‡

Leader + GPI Leader Non leader +GPI

12.

1 2 3 4 5 6 7 8 9 10 Af293

715 bp

Afu3g08990Score=432

1035bp

1001 bp

Afu6g14090 CFEMScore=134690bp

Afu2g05150 (MP-2)Score=301

Afu4g09600 Score=292

Strain:

A.fumigatus GPI anchored geneswith high ETANDEM scores have

size-variable repeats

13.

Top-scoring A. fumigatus GPI-anchoredTR genes transcribe

size-variable mRNA

Afu6g14090

Afu3g08990

Afu4g09600

690 bp

1031 bp

724 bp

Afu2g051501001 bp

1 2 3 4 5 6 7 8 9 10 Af293 Expected

14.

GPI-anchored TR genes encode for

discrete numbers of in-frame repeats

15.

Cons. Hypoth. CFEM

Cons. Hypoth.

MP-2 immunogenic secreted.

Cons. Hypoth.

Top scoring GPI & leader-Top scoring GPI & leader-containing genes at containing genes at different time pointsdifferent time points

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Afu2g05150 Afu3g08990 Afu4g09600 Afu6g14090

time (hrs)

expr

essi

on fo

ld-in

crea

se

0 hr 6 hrs 12 hrs 20 hrs

Δ ΔΔ

16.

Afu3g08990 in silico analysis

(~PGQPSV)18-32 six aa C-terminal rpts

Extended rod domain of collagen

MVNSGIALLALAGPALGGIVPNKGNDEGHSGYPGHTPPTKWPATAEPHWVTRTATYLTTTCPVSHTKVTSGTKTLTIPITLTSTITVTTTTVEDSNTETRVVVPTGTVPNHPLVPTSVPTSVPTSVPTSVPTQNATSIPTFVPTQNATSVPTSVPTQNATTIVPPPKVVPTGVPGQNTTVVPPPRPAPFTNSTVPPPVQPVPVAPGQPTAPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAHEQPSVPGQPSVHEQPSDPEQPSVPGQPTAVSPPPAETALTGSASQFEPAAGLLAGIWVVMLML

P/T/S rich N-terminal region

17.

tBlastN fungal E-value

gb|AAHF01000002.1| Aspergillus fumigatus 00.ref|NZ_AAKE03000010.1| Neosartorya fischeri 9e-26ref|NZ_AAKD03000018.1| Aspergillus clavatus 6e-09gb|AACD01000051.1| Aspergillus nidulans 3e-06gb|AAJN01000190.1| Aspergillus terreus 8e-04gb|AAIH01000116.1| Aspergillus flavus 8e-04ref|NZ_AAFU01001164.1| Chaetomium globosum 0.002ref|NC_005785.3| Ashbya gossypii 0.011gb|AAEG01000002.1| Saccharomyces cerevisiae 0.041

Afu3g08990-like genes are Aspergillus-specific and extremely

divergent

18.

MVNSGIALLALAGPVFAGIMPNKGNDEGHPGYPGPTPPTKWAATPEPHWVTREITYLTTSCPISHTKVTSGTKTLTVPITLTNTITVTTTTVHDLNTETRVVVPTGTATTRPLVPTSVPTSVPTQNATTIVPPPPKIVPTGVPGQNTTVIPPPRPAPFTNSTVPPPVQPVPVAPGQPSAPSQPSVPGQPSVPGQPSVPSQPSVPSQPSVPGQPSVPSQPSVPGQPSVPQQPSVPQQPSVPGEPTVVSPPPAETSLTGSASQFGPAAGLLAGIWVFM LML

MVKAGVALVAFAGPAFAGVIGTGSHDQVSLLAQACPSCSIMLTRENSKPHVGPTAPIKPPSNSEKWTTTTEPHWVTKTTTFVTTSCPVTHSTVTSGTKTLTVPITLTETVTLTTTTVCDLNTEPPEVIVPTGVVTTKPLVPTSVTTTETVCTTCHHNNTTVPPHSSTYVPGHNTTIVPPHSTTQVPGHNTTIVPPHSTTQIPGHNTTIVPPGTTNGTIVVPPTSVPSHNTTVIPPAPVPTQNPGQNNTVVPIPPKPAPFTNSTVPSVPTVQPVPVPPVVPGQPSVPGQPSVPGQPSIPGQPSTPGQPSTPGQPSLPGQPGVPEQPSTPGQPSVPGQPSTPGQPSTPGQPSTPGQPSTPGQPSTPGQPSTPGQPSTPGQPSTPGQPSTPGQPSAPGQPSVPGQPSTPGQPGVPEQPSTPGQPSTPGQPSTPGQPSVPGQPGVPEQPSTPGQPGVPEHPSTPGQPAAPGESSVPGHPETPAPSASPAQPSGVSPVSPSESSHPFTGSAAQSFKPAAGLLAGVMGLMLLL

MVNKIVALLALTTPAFAGVAHWKEPHWKEPHDKEPHSVSPGGQLRPAQTTTYTTTTCPVTSTVVTSGTKTLTVPITLTNTVTITTTYCEDETRPPVHPTAPTTGKPAPPVTGNPGPTGTPAPTGKPVPTGKPTPIPTGKPRPVPTGPVNNGTAPPNPPFQNSTSPPLIPTTRPVPVPPGSSPAPVPVPVPVPSPGQPEQPGTPAAPGGSATPSIPGQPGTPAAPGGSGHPVTPGQPGQSSQPGQPGKPGLPGQPGQSGQPGQPGQPGQSGQPGQPGQPGQSGQPGQPGQPGQSGQPGQPGQPGQPGPSGTPAAPGGSSNPVIPGKPGTPGASGVPSTPSAPGASGTPGVPGAPHASSTPSVPTAPGASGTPEAPAQPGKPDQPGKPGHPGEVSPEAPPTGVSPARPDFTGSADNVRPAAGLLIGLMTIMALL

MRYSTSVVTALALGRRCSCCRAQQAWPQVGHSYGLLPLPLKSLPLRLSPLPPAEKPSAPAQPSAPAEKPSAPAQPSAPAEKPSAPAQPSAPAQPSAPAQPSAPAQPSAPAQPSAPAQPSAPAQPSAPAQSSAPAQPSKATSVPVIPAPVSSAPVSSAAVPSSKAASSSVLVSSQKPKSTSSATSTGTNTVGGNEGGVAPPAQTSIGAANPGSTIYVPGVAMAGSIVMGLMVLA

A. fumigatus Afu3g08990

Neosartorya fischeri 9e-26

Aspergillus clavatus 6e-09

Aspergillus nidulans 3e-06

Aspergillus terreus 8e-04

MVNSGIALLALAGPALGGIVPNKGNDEGHSGYPGHTPPTKWPATAEPHWVTRTATYLTTTCPVSHTKVTSGTKTLTIPITLTSTITVTTTTVEDSNTETRVVVPTGTVPNHPLVPTSVPTSVPTSVPTSVPTQNATSIPTFVPTQNATSVPTSVPTQNATTIVPPPKVVPTGVPGQNTTVVPPPRPAPFTNSTVPPPVQPVPVAPGQPTAPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAHEQPSVPGQPSVHEQPSDPEQPSVPGQPTAVSPPPAETALTGSASQFEPAAGLLAGIWVVMLML

19.

Lactobacillus hyp. 10-321562 aa

1369 aa Tetrahymena FH2 10-27

1212 aa Strep. agalactiae 10-27C-protein β-ag

940 aa Tryp. cruzi trans-sialidase 10-20

430 aa Afu3g08990

TR region

954 aa Human collagen type XXI 10-17

Pan-species Blast reveals little.…20.

0

0.5

1

1.5

2

2.5

Afu3g08990

AMB CAS ITZ β-Glucanase

CR H2O2 Calcofluor SDS

Fold

expr

essio

n

Un-treated

0

0.5

1

1.5

2

2.5

Afu3g08990

AMB CAS ITZ β-Glucanase

CR H2O2 Calcofluor SDS

Fold

expr

essio

n

Un-treated

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

membrane

21.

0

20

40

60

80

100

4 6 8 12

∆3

∆1∆2

randomAF 293KI

Time (h)

Hyp

hal

len

gth

in

µm

Deletion of ∆Afu3g08990 leads to early germination

22.

AF 293 ∆1 ∆2 ∆3 random KI0

20

40

60

80

100

120

140

Con

idia

per

fi

eld

The ∆Afu3g08990 strains exhibit reduced adhesion to

ECM

23.

Afu3g08990 repeat region can be used for strain typing

Balajee et al. 2007, submitted.

Summary Summary Afu3g08990Afu3g08990

•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)

24.

What does What does Afu3g08990Afu3g08990 dodo??

1 .Receptor?

2 .Cross-linker ‘cement? ’

25.

PM

3 .Reservoir model

Yana Shadkchan

Emma Levdansky Haim SharonKobi Romano

26.

Kevin Verstrepen, Harvard

Gerry Fink, Whitehead Inst.

Collaborators:

Thank youThank you! ! 28.