TB Symposium, Barcelo Hotel September 20-22

Transcription

1 Evolution of Mycobacterium tuberculosis PE/PPE multigenefamilies By Helmi Mardassi, Institut Pasteur de Tunis TB Symposium, Barcelo Hotel September 20-22

2 PE/PPE multigene families have been uncovered owing to the availability of the M. tuberculosis genome sequence PE mulitigene family PPE multigene family (S.Cole et al., 1998)

3 Structure and sequence of the PE_PGRS33 member (M. tuberculosis Rv1818c) (Delogu and Brennan, 2002)

4 Evidence for a role of PE/PPE genes in host-cell interaction A transposon mutant of PPE 46 was found to be attenuated for growth in macrophages (Camacho et al., ) 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 31,, PPE68 68,, 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

5 Putative roles 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

6 Duplication of PE/PPE occurred in association with the ESAT-6 (esx) gene cluster (Gey van Pittius,2006)

7 Structural genomics/structural biology determined the crystal structure of a PE/PPE protein complex Strong et al., 2006

8 A plausible scenario for the expansion of the PE/PPE gene families has been proposed

9 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)

10 Molecular mechanisms that might operate under natural conditions to generate 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 (Namouchi et al., 2008, Gey van Pittius et al., 2009)

11 EVIDENCE FOR GENE CONVERSION RECOMBINATION

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13 PE_PGRS16 RpmF Rv0981 Rv0982 FadE13 Rv0976c Rv0979c PE_PGRS17 PE_PGRS18 12/40 polymorphism PE_PGRS17 PE_PGRS18 M. tb H37Rv and H37Ra MAR1 MAR2 MAR1 MAR2 PGRST3 M. tb CDC1551 PGRST2 M. tb 210 and M. bovis AF2122/97 PGRST1 PE PGRS PE PGRS

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16 The 12/40 polymorphism was not randomly distributed PGRST1 PGRST1>PGRST2>PGRST3 PGRST1 PGRST1 PGRST1

17 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

18 Within the whole collection of MTBC strains, only the three newly defined PGRST types could be identified PE_PGRS17 PE_PGRS18 PGRST1 (+/-) PGRST2 (+/+) PGRST3 (-/-)

19 The gene conversion event occurs independently multiple times

20 The 12/40 polymorphism could represent an evolutionary marker to trace back the MTBC progenitor and other smooth tubercle bacilii (-/-)

21 Conclusion 1 The 12/40 polymorphism provided evidence for the occurrence of gene conversion in the natural evolution of the MTBC 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

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23 Throughout the mycobacterial genome, PE/PPE genes occur mainly in the context of loci

24 Criteria used for the design of oligoprobes The oligonucleotides were selected using the public-domain software, OligoPicker. The 50 mers oligonucleotides were selected from the known list of PE/PPE genes present in M. tuberculosis, M. bovis, and M. avium subspecies paratuberculosis. The parameters used were: - a maximum continuous match to non-target sequences of 16 nucleotides and a range Tm of 10 degrees C. -those that showed more than 50% sequence identity with another genomic location were discarded, -those that failed to discriminate between the test strains H37Rv, CDC1551 and M. bovis were eliminated.

25 Hybridization signals were tested for specificity using reference strains

26 Six LSPs (LSPTun1 to LSPTun6) were identified throughout 33 Tunisian isolates belonging to representative genotypes

27 Abdallah et al Mol. Microbiol ESX-5

28 LSPTun2 LSPTun4 LSPTun5 LSPTun6

29 CONCLUSION 2 As surface-exposed and immunodominant genes, PE/PPE exploits homologous recombination to diversify their antigenic repertoire as well as their expression pattern, and hence, to adapt to environmental changes/escape from the host immune system

30 CONCLUSION Gene conversion contributes to variability of PE genes PE genes are subjected to both intragenic and intergenic recombination not only to diversify their gene sequence and acquire new antigenic properties, but also to modulate their expression pattern. In MTBC, the data showed that PE/PE_PGRS genes are prone to extensive genetic variability in STB. a finding in line with their involvement in many aspects of hostpathogen interaction and their particular expansion in pathogenic mycobacteria.

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32 Acknoweldgements Dr. Anis Karbojul Dr. Amine Namouchi Melle Leila Jeljeli Nico Gey van Pittius Cristina Gutierrrez Thank you all!