Putative Biology of PEPPE Multigene Families of Mycobacteria and their Relevance with Regard

1

• Putative Biology of PE/PPE Multigene Families of
  Mycobacteria and their Relevance with Regard
• to Evolution
• Helmi Mardassi, Institut Pasteur de Tunis

2
M. tuberculosis, the most successful human
pathogen, displays restricted genetic
polymorphism and appears to be exceptionally
stable
H37Rv, H37Ra
LAM , Haarlem
Beijing
Sreevatsan et al., 1997
3
PE/PPE gene families have been uncovered owing to
the availability of the M. tuberculosis genome
sequence
(S.Cole et al., 1998)
4
Structure of the PE family members (99 members)
5
Structure and sequence of the PE_PGRS33 member
(M. tuberculosis Rv1818c)
6
The PPE family (69 members)
7
Constraints 1. Repetitive sequences extensive
cross reactivity (hybridization and antigenic
assays) 2. high GC content very difficult to
amplify by PCR 3. Poorly expressed in the
conventional host E.coli (Toxic effect?
Instability?) 4. Very acidic and
membrane-associated proteins notoriously
refractory to analysis by two-dimensional
electrophoresis and mass spectrometry
8
PE/PPE genes are particularly abundant within the
M. tuberculosis complex
9
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10
A plausible scenario for the expansion of the
PE/PPE gene families has been recently proposed
11
Several questions relating to these multigene
families came to mind 1. Do all PE and PE_PGRS
proteins share similar functions? 2. What is the
extent of their genetic variability? 3. Do they
really play a role in antigenic
variations 4. Are they associated to enhanced
virulence and /or transmissbility? 5. Which
members are conserved and essential
12
Two functions were immediately proposed
(Cole et al., 1998)
13
The PGRS domain of PE_PGRS proteins dispalys
significant sequence similarity with the EBNA1
antigen of EBV
(Brennan Delogu, 2002)
14
The PGRS domain confers increased stability to
GFP protein in eucaryotic cells
(Brennan Delogu, 2002)
15
Michael Brennan
PGRS33 DNA vaccine reasonable level of
protection Vaccine produced ab response to only
PGRS tail region and not PE PE region high
Inf-? (low IL-10, no ab) high Th1 response PGRS
region low Inf-? (high IL-10, high ab) high
Th2 response
16
PE/PPE are genetically variable
? In silico comparative sequence analysis (Cole
et al., 1998, Gordon et al., 2001, Fleishmann et
al., 2002, Garnier et al., 2003) ? Sequence
analysis of clinical isolates PE_PGRS33
(Talarico et al., 2005), PPE8 (Srivastava et al.,
2006, PE_PGRS17, PE_PGRS18 (Karboul et al.,
2006) ? Microarray data (Tsolaki et al.,
2004 Garcia-Pelayo et al., 2004)
17
The Molecular mechanisms that operate to generate
the genetic variability in PE/PPE genes
? Dislocations between a replicating strand and
its template at repetitive DNA sequences
(replication slippage) (Cole et al., 1998,
Machowski et al., 2007)
? Intergenic and intragenic recombiantion/gene
conversion events (Cole et al., 1998, Gutacker et
al. 2006, Karboul et al., 2006, Lui et al., 2006)
? Microsatellite polymorphism (Sreenu et al.,
2006)
? Insertion deletion events of IS and phage
sequences within PE/PPE genes
18
A genomic library-based amplification strategy
(GL-PCR) for efficient mapping of insertion
sequences
19
A typical GL-PCR profile
20
Rv2815c Rv2816c
Rv2328 PE23
Rv1755 plcD
Rv0403c mmpS1
Rv0794cRv0795c
Rv2819c
Rv2017Rv2018
Rv2352c PPE38
Rv0171 mce1c
Rv2336
Genomic location of IS6110 in the M. tuberculosis
reference strain MTB14323
21
PE/PPE genes are differentially expressed
? DFI
? Promoter trap
? cDNA microarray
? RT-PCR and QRT-PCR
22
Subcellular location
? PE/PPE proteins are associated with the cell
wall and cell membrane fractions and appears to
be partly exposed on the cell surface (Doran et
al., 1992 Brennan et al., 2001 Banu et al.,
2002, Sampson et al., 2001 Okkels et al., 2003
Delogu et al., 2004 Le Moine et al., 2005)
? In silico analysis identified in 40 PE/PPE
proteins potential beta-barrel outer-membrane
structures (Pajon et al., 2006)
? PE_PGRS33 influences the cellular architecture,
colony morphology and bacillus-bacillus
interaction (Brennan et al., 2001)
23
Structural genomics/structural biology determined
the crystal structure of a PE/PPE protein complex
Strong et al., 2006
24
Additional putative function(s) and relevance of
the PE/PPE proteins
1. Antigenic variations 2. Interference with
antigen processing and presentation 3. Necessary
for replication and persistence of the bacillus
within the host cell 4. Vaccine
candidate 5. Pre-clinical expression
diagnostic potential (in the mouse model)
6. Architecture of the bacillus
25
Phenotypic characteristics as inferred from gene
inactivation based experiments

• ? A transposon mutant of PPE 46 was found to be
  attenuated for growth in macrophages (Camacho et
  al.,1999)
• ? In the M. marinum model, two PE_PGRS genes were
  found to be essential for the bacillus to
  replicate in macrophages and persists in the host
  granulomas (Ramakrishnan et al., 2000)
• ? M. Bovis BCG strain, whose PE_PGRS33 expression
  is abrogated could not infect and survive in
  macrophages (Brennan et al., 2001)
• ? PPE31, PPE68, and PE35 are required for growth
  in vivo during infection of mice (Sassetti et
  al., 2003 )
• ? PPE25 ( Li et al., 2005) and PPE10 (Stewart et
  al., 2005) mutants seem to be associated with the
  control of phagosomal acidification

26
Effect of expression of certain PE_PGRS genes in
the non pathogenic M. smegmatis
? M. Smegmatis expressing PE_PGRS33 displayed
enhanced colonization of BMM macropahages and
increased cell necrosis (Dheenadhayalan et al.,
2005) ? PE_PGRS33 elicits TNF-alpha release
from macrophages in a TLR2-dependent manner (Basu
et al., 2007) ? M. smegmatis expressing the
PE_PGRS gene Rv3812c display increased resistance
in vitro to low pH (Karboul et al., in
preparation)
27
CONCLUSION
?Expansion of PE/PPE proteins in pathogenic
mycobacteria seem to have been accompanied with
functional divergence ? Although several members
are homologous, there does not seem to be any
compensatory effect. Thus, a high level of
functional specialization could have been reached
during evolution ? From these preliminary
studies, certain members seem to be endowed with
multiple functions ? Although deletion analyses
of PE/PPE genes were accompanied with phenotypic
characteristics, the detailed molecular
mechanisms responsible for the observed effects
remain to be demonstrated
28
The research program relating to PE/PPE gene
families carried out at IPT
Primary objective Evaluate the distribution of
PE/PPE genes among mycobacteria and the extent of
their genetic variability
Specific aims -Comparative sequence analyses of
selected genes -Development of efficient tools
for the specific detection of PE/PPE genes
(identification of specific probes)
29
PE34
Rv3872
Rv3020c
Rv1089
Rv3893c
Rv0335c
Rv3018A
Rv3022A
Rv0285
Rv1386
PE/PE_PGRS members subjected to comparative
sequence analysis
Rv2408
Rv2328
Rv3812
Rv1646
Rv2107
Rv1169c
Rv3097c
Rv2431c
Rv0151c
Rv0152c
Rv0159c
Gr.1
Rv0160c
Rv1430
Rv3650
Rv1172c
Rv2099c
Rv1788
Rv1791
Rv2769c
Rv1040c
Rv3622c
Rv1195
Rv3477
Rv0754
Rv1806
Rv2340c
Rv0916c
Rv3652
Rv1088
Rv1983
Rv2519
Rv1214c
Rv0977
Rv0109
Rv1768
Rv1087
Rv2162c
Rv1091
Rv1840c
Rv1651c
Rv3653
Rv2098c
Rv1803c
Rv0532
Rv0124
Gr.2
Rv1396c
Rv0578c
Rv1067c
Rv1068c
Rv1468c
Rv3388
Rv0278c
Rv0279c
Rv0747
Rv0742
Rv2741
Rv1818c
Gr.3
Rv0746
Rv2396
Rv1325c
Rv0834c
Rv2591
Rv3344c
Rv3512
Rv0833
Rv2126c
Rv3507
Rv0297
Rv1243c
Rv2490c
Gr.4
Rv0872c
Rv2853
Rv2487c
Rv3367
Rv3595c
Rv0832
Rv3590c
Rv2634c
Rv1441c
Rv3345c
Rv0978c
Rv0980c
Gr.5
Rv2371
Rv2615c
Rv1450c
Rv1452c
Rv3508
Rv3514
Rv3511
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PCR amplification of PE members through the
Mycobacterium tuberculosis complex
Rv 0978
Rv 0285
Rv 0160
Rv 1169
Rv 3367
Rv 1195
Rv 0980
Rv 1040
Rv 1441
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Sequence analysis of 22 PE members in
Mycobacterium tuberculosis complex RESULTS
0 PE 5 PE_PGRS
4 PE_PGRS 10 PE
1 PE 2 PE_PGRS
32
Among the highly variable genes, two conform with
the definition of a duplicated gene pair
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  PE_PGRS17 250
260 270 280 290 300
310 320
........................................
........................
Q A G S T Y A V A E A A S A T P
L Q N V L D A H37Rv and H37Ra
CAAGCTGGCAGCACCTACGCGGTCGCCGAAGCGGCCAGCGCAACACC
GCTGCAGAA------------CGTGCTCGATGC CDC1551
.............................................
.........C.GATCGAGCAGGC.C..T.G.GG.T M.bov
AF2122/97 ....................................
..................C.GATCGAGCAGGC.C..T.G.GG.T

Q I E Q A L G V


330 340 350 360 370
380 390 400
........................................
........................
I N A P V Q S L T G R P L I G
D G A N G I D G T G Q H37Rv and H37Ra
GATCAACGCACCCGTTCAGTCGCTGACCGGGCGCCCATTGATCGGC
GACGGCGCGAACGGGATCGACGGGACCGGGCAAG CDC1551
.......A.G..GAC.G..G.....GTG......AAGC......
.T........CC.....GCGCC...C........G. M.bov
AF2122/97 .......A.G..GAC.G..G.....GTG......AA
GC.......T........CC.....GCGCC...C........G.
T T E A V
K H A P  
410 420 430 440
450
........................................
A G G N G G W L W G
N G G N G G S H37Rv and H37Ra
CCGGCGGTAACGGCGGGTGGCTGTGGGGCAACGGCGGCAACGGCGGGTCG
CDC1551 .......GGC......CATCT........
...........T......... M.bov AF2122/97
.......GGC......CATCT...................T.........
A I
34
PE_PGRS18   250
260 270 280 290 300
310 320
........................................
........................
Q A G S T Y A V A E A A S A T P
L Q N V L D A H37Rv and H37Ra
CAAGCTAGCAGCACCTACGCGGTCGCCGAAGCGGCCAGCGCAACACCG
CTGCAGAA------------CGTGCTCGATGC CDC1551
..............................................
........C.GATCGAGCAGGC.C..T.G.GG.T M.bov
AF2122/97 ....................................
....................------------............

Q I E Q A L G V  
330 340
350 360 370 380 390
400 ................
........................................
........ I N A P V
Q S L T G R P L I G D G A N G I D
G T G Q H37Rv and H37Ra
GATCAACGCACCCGTTCAGTCGCTGACCGGGCGCCCATTGATCGGCGACG
GCGCGAACGGGATCGACGGGACCGGGCAAG CDC1551
.......A.G..GAC.G..G.....GTG......AAGC.......T..
.C....CC.....GCGCC...C........G. M.bov AF2122/97
..............................................
..................................
T T E A V K
R H A P
410 420 430 440 450
................
........................
A G G N G G W L W G N G G N G G
S H37Rv and H37Ra CCGGCGGTAACGGCGGGTGGCTGTGG
GGCAACGGCGGCAACGGCGGGTCG CDC1551
.......GGC......CATCT...................T.........
M.bov AF2122/97 ............................
......................
A I
35
The distribution of the 12/40 polymorphism could
define three new PE_PGRS-based groups
PGRST3
PGRST2
PGRST1
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Development of a reverse hybridization assay
(PEGAssay) for the large scale analysis of the
12/40 polymorphism distribution
18
17
M.
tuberculosis
(H37Rv)
M.
tuberculosis
(H37Ra)
Negative
control (
Buffer
)
M.
tuberculosis
(
Erdman
)
M.
tuberculosis
(CDC1551)
M.
smegmatis
(
mc
²
155)
M.
africanum
(ATCC 25420 )
M.
microti
(ATCC 35782)
M.
pinnipedii
(FCC69)
M.
caprae
(CIP 105776)
(AF2122/97)
M.
bovis
M.
bovis
BCG
(A
TCC 27290)
41
Overall, 521 MTBC isolates were analyzed
415 M. tuberculosis 108 PGG1(57 Ancestral), 259
PGG2, 48 PGG3 42 M. bovis (5 BCG
strains) 30 M. africanum (14 A1, 6 A2, 8
A3) 17 M. microti (9 voles, 3 llama, 2 cat, 2
human, 1 pig) 3 dassie 4 M. pinnipedii 2 M.
caprae 6 M. canettii and 2 smooth tubercle
bacilli
42
Within the whole collection of MTBC strains, only
the three newly defined PGRST types could be
identified
PE_PGRS17
PE_PGRS18
PGRST1 (/-)
PGRST2 (/)
PGRST3 (-/-)
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Gene conversion is a class of homologous
recombination
Recombinant DNA
Gene conversion
Parental DNAs
Crossing over
Recombinant DNAs
46
Strand break coupled to mismatch repair as the
most plausible explanation for gene conversion

-


47
The gene conversion event occurs independently
multiple times
48
CONCLUSION

• As far as could be ascertained, this is work
  provided the most obvious gene conversion event
  in the natural evolution of the mycobacterial
  species

• The findings reinforce the role of gene
  conversion as a mechanism for the generation of
  genetic variability associated with PE/PPE
  families

• Strains of the M. bovis lineage appear to be
  refractory to gene conversion

49

• The study offers a new perspective to trace back
  the evolution of tubercle bacilli

and other smooth tubercle bacilii (-/-)
50
Phylogenetic analysis of smooth tubercle bacilli
(referred to as M. prototuberculosis) provided
insights into the genetics of strains that might
have predominated prior to the expansion of the
MTBC
Gutierrez et al., 2005
51
The sequence polymorphism within the housekeeping
genes of the smooth tubercle bacilli group shows
gene mosaicism
52
Genetic variability of the PE_PGRS duplicated
genes
53
The duplicated PE_PGRS members were previously
shown to be preferentially upregulated in vivo
54
Development of efficient tools for the specific
detection of PE/PPE genes
55
Development of a perl scripting program for the
identification of PE/PE_PGRS member specific
sequence
ATCGGGATCCAGGAATTCGATCCCCGGTTTTAACTATACGCATGTCATGC
AAGTCCCGTGGGGG
PE/PE_PGRS database
Script 1 extracts a specified length of the
sequence from the gene sequence starting from the
first and shift to the second and so on until the
last possible sequence of the desired length is
obtained. This constitute the candidate primers
30-base window size
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
CCTTAAGGTTGCAACACATGTGGGCCTTAGGAGTCGTTGTTTGTTACGTA
ATGGGCGTTGG
56
Script 2 converts a sequence in FASTA format to a
format that enable the candidate primers to
search the gene sequence
Candidate primers database
Script 3 search for number of times the
candidate primer occurs in the gene sequence.
Extract the one which occur once.
Putative PE/PE_PGRS member specific primer
57
30 mer signature sequences (100 identity)  PE
subfamily PE30_175_204 catggtcaggactatcaagct
cttagcgca Rv3097c PE30_176_205
atggtcaggactatcaagctcttagcgcac Rv3097c
PE30_177_206 tggtcaggactatcaagctcttagcgcaca
Rv3097c PE30_178_207 ggtcaggactatcaagctctt
agcgcacag Rv3097c PE30_179_208
gtcaggactatcaagctcttagcgcacagc Rv3097c
PE30_180_209 tcaggactatcaagctcttagcgcacagct
Rv3097c PE30_181_210 caggactatcaagctcttagc
gcacagctt Rv3097c PE22_46_75
gcgacactggagtcccttggttcccacatg Rv2107
PE22_47_76 cgacactggagtcccttggttcccacatgg
Rv2107 PE22_48_77 gacactggagtcccttggttcc
cacatggc Rv2107 PE22_49_78
acactggagtcccttggttcccacatggcg
Rv2107 PE2_776_805 ttgcaggcatcacattcgtacaca
ccaagt Rv0152c PE2_777_806
tgcaggcatcacattcgtacacaccaagta Rv0152c
PE2_778_807 gcaggcatcacattcgtacacaccaagtat
Rv0152c PE2_779_808 caggcatcacattcgtacaca
ccaagtatt Rv0152c PE26_1290_1319
tatctcaatctcaatacatgacaaccagac Rv2519
PE26_1288_1317 gttatctcaatctcaatacatgacaaccag
Rv2519 PE26_1289_1318 ttatctcaatctcaatacatga
caaccaga Rv2519 PE4_862_891
ccggcgaatagtccctacccgacacacatt
Rv0160c PE12_242_271 tgagagcaagtgcagacgcgtatg
caaccg Rv1172c PE18_229_258
gtcaacactctacagatgagctcagggtcg Rv1788
PE1_1209_1238 cgaaccgaacttggaagtaatcgtcaatct
Rv0151c PE24_134_163 caattgccgcaatattgctgt
cacacgccc Rv2408  
58
PE_PGRS subfamily PE_PGRS21_1027_1056
gtcaccttcagtagtagcttaagtggcctt Rv1087
PE_PGRS21_1028_1057 tcaccttcagtagtagcttaagtgg
ccttt Rv1087 PE_PGRS21_1029_1058
caccttcagtagtagcttaagtggcctttc Rv1087
PE_PGRS21_1030_1059 accttcagtagtagcttaagtggcc
tttcc Rv1087 PE_PGRS21_1031_1060
ccttcagtagtagcttaagtggcctttccg
Rv1087 PE_PGRS21_1032_1061 cttcagtagtagcttaagt
ggcctttccgg Rv1087 PE_PGRS62_585_614
ggcgtactacatccaacagattattagctc
Rv3812 PE_PGRS62_586_615 gcgtactacatccaacaga
ttattagctcg Rv3812 PE_PGRS62_587_616
cgtactacatccaacagattattagctcgc Rv3812
PE_PGRS62_588_617 gtactacatccaacagattattagc
tcgca Rv3812 PE_PGRS62_589_618
tactacatccaacagattattagctcgcag Rv3812
PE_PGRS62_590_619 actacatccaacagattattagctc
gcaga Rv3812 PE_PGRS62_591_620
ctacatccaacagattattagctcgcagat Rv3812
Using a window size of 30 mers 9 PE member
specific sequences 2 PE_PGRS member specific
sequences
59
Results of the probe search
Overall 2187 member specific sequences targeting
PE and PPE families were derived
60
Hybridization of a biotinylated PCR product
corresponding to a PE_PGRS gene with member
specific sequences of 34 other PE/PE_PGRS gene
probes
61
Set up of a 50 mer-based PE/PPE specific
microarray protocol
Initial conventional hybridization conditions
Improved hybridization conditions
62
29 polymorphic sites out of a total of 177 could
be detected
58 Tunisian isolates
Controls
M. bov
M. bov
H37Rv
CDC1551
63
Towards PE/PPE-based phylogenomics
64
Acknowledgements Part of this work was supported
by funds from the United Nations Development
Program/World Bank/World Health Organization
Special Program for Research and Training in
Tropical Diseases (TDR). Special thanks to Anis
Karboul and Amine Namouchi, Nico Gey van Pittius
(US, Cape Town), Roland Brousseau (BRI,
Montreal), and Cristina Gutierrez (IP, Paris).