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Interspecies horizontal gene transfer (HGT)

Interspecies horizontal gene transfer (HGT). Important in the evolution and virulence of Mycobacterium tuberculosis. Horizontal Transfer of a Virulence Operon to the Ancestor of Mycobacterium tuberculosis. Bayona, Sarah Jane de Guzman, Brian James. Introduction. Objectives.

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Interspecies horizontal gene transfer (HGT)

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  1. Interspecies horizontal gene transfer (HGT) • Important in the evolution and virulence of Mycobacterium tuberculosis

  2. Horizontal Transferof a Virulence Operonto the Ancestor ofMycobacterium tuberculosis Bayona, Sarah Jane de Guzman, Brian James

  3. Introduction

  4. Objectives • To be able to study evolutionary history of the M. tuberculosis Rv0986-8 virulence operon through functional genomics approaches, as playing an important role in parasitism of host phagocytic cells. • To be able to show the contribution of HGT to the emergence of M. tuberculosis and close relatives as major pathogens • To be able to study that among actinobacteria, this operon is specific to the M. tuberculosis complex and to ancestral Mycobacterium prototuberculosis species • To be able to determine if this operon has been aquired horizontally by the ancestor of M. tuberculosis, before the recent evolutionary bottleneck that preceded the clonal-like evolution of the M. tuberculosis complex • To be able to determine if the transfer was plasmid mediated and if the operon originated from a c-proteobacterium donor species using genomic signature profiling.

  5. Horizontal gene transfer (HGT) between unrelated species • additional factor driving genetic diversity, especially in bacteria • provides the host organism with new genes that under certain selective pressures may prove to confer a selective advantage and therefore may be maintained and/or expanded within the population. • pathogenicity islands  virulence genes and gene clusters acquired by HGT pathogenic bacteria (not common in mycobacteria) observed in fast and slow-growing mycobacteria species, indicating that they likely represent ancient episodes of HGT occurred in an ancestor of the whole Mycobacterium genus.

  6. Whole-genome analysis of the chromosomes of Mycobacterium tuberculosis and of Mycobacterium bovis • did not allow identifying any large DNA fragment with aberrant GC content, a genetic signature of DNA acquired through HGT, including pathogenicity island • consistent with the natural reluctance of mycobacteria to exchange genetic material

  7. Recent genetic study of ancestral Mycobacterium prototuberculosis • many intraspecies HGT events must have occurred in the progenitor of M. tuberculosis before the evolutionary bottleneck leading to emergence of the M. tuberculosis complex possibly from the Indian subcontinent

  8. M. tuberculosis virulence genes • Identified through signature-tagged transposon mutagenesis involved in macrophage parasitism • Involved in one or more steps of host cell infection including • attachment to the cell surface, • cell entry, • intracellular trafficking, • inhibition of phagosome-lysosome fusion, and • intracellular bacterial metabolism.

  9. M. tuberculosis Rv0986 • Forms an operon with Rv0987 and Rv0988 and encodes an ATP-binding cassette (ABC) transporter involved in early interactions between the bacillus and host cells • Mycobacterium tuberculosis mutants inactivated in Rv0986 and Rv0987 show reduced ability to bind to macrophages and to inhibit phagosome-lysosome fusion in vitro

  10. Methods employed • Mycobacteria Culture and Genomic DNA Extraction • Polymerase Chain Reaction Amplification and Sequencing • Southern Blotting Analysis • Bioinformatics

  11. Results and Discussion

  12. Mycobacterium tuberculosis Rv0986-8 is Orthologous to A. tumefaciensattE–H • Figure 1.A. Genetic organization of the M. tuberculosis Rv0986-8 gene cluster, as compared to that of Agrobacterium tumefaciens attE–H operon. Percentages indicate similarity between proteins. Because Agrobacterium and Mycobacterium are two phylogenetically distant genera, evolutionary history of the Rv096-8 operon was investigated in more detail.

  13. Rv0986-8 is Specific to the M. tuberculosis Complex and Ancestral M. prototuberculosis Species Among Actinobacteria Figure 1.C. Southern blot analysis of BamHI-digested genomic DNA from various mycobacteria using radiolabeled Rv0988 as a probe. Figure 1.B. PCR analysis of genomic DNA from species of the M. tuberculosis complex using oligonucleotides in the Rv0986-8 cluster-flanking genes mscL and grcC1 strongly suggested that this operon was specific to the M. tuberculosis complex, including M. tuberculosis, M. bovis, Mycobacterium africanum, and Mycobacterium microti

  14. Figure 1.D. Genetic organization of the Rv0986-8 orthologous region in Corynebacterium glutamicum and in various fast- and slow-growing mycobacteria, as (1) retrieved from the complete genome sequences or (2) infered from (B) and (C). Pluses and blanks indicate the presence and absence of orthologues, respectively.

  15. Figure 2.A Protein tree of Rv0988. Blue, red, green, and purple colors indicate b-, c-, a-, and other proteobacteria, respectively.

  16. Figure 2.B The part of the tree within dashed line in (A) is shown at higher magnification in (B). Ps, Pseudomonas syringae; Pf, Pseudomonas fluorescens; Rp, Pseudomonas palustris; Bj, Bradyrhizobiumjaponicum; Pp,Pseudomonasputida;S, Silicibacter sp; Rs, Rhodobacter sphaeroides; Sp, Silicibacter pomeroyi.

  17. Figure 3.A Rv0986-8 derives from HGT. (A) GC content of the Mycobacterium tuberculosis 1096.5–1013.0 kb genomic region. The dashed line indicates the global GC content of the H37Rv genome (65.6%).

  18. Figure 3.B Comparison of codon usage for leucine (LEU), isoleucine (ILE), and lysine (LYS) in Rv0986-8 and in the M. tuberculosis H37Rv whole coding genome.

  19. Figure 3C. The 3:1 dinucleotide analysis of mprA-mscL (5 to 1), Rv0986-8, and grcC2-Rv0992c (11 to 14).

  20. Figure 4A. Rv0986-8 gene cluster possibly derives from plasmidmediated transfer from c-proteobacteria. (A) Four-letter word genomic signature of the Rv0986-8 gene cluster (left panel), of Mycobacterium tuberculosis H37Rv whole genome (middle panel), and of the c-proteobacterium species, Alcanivorax borkumensis (right panel), at the closest genomic distance from Rv0986-8.

  21. Figure 4 B. Distance tree between genomic signatures of Rv0986-8 gene cluster and the 32 closest signatures among ;20,000 species.

  22. Conclusion

  23. Our results strongly support the hypothesis that the ancestor of the M. tuberculosis complex may have been, like Mycobacterium marinum and most mycobacteria, an early environmental species that subsequently colonized eukaryotes. • The acquisition of Rv0986-8 and of other genes presumably conferred a selective advantage to this ancestor, making it possible to colonize plants or lower animals, such as prototype M/s (amoebae).

  24. These genes may have been subsequently used by species of the complex to facilitate the colonization of other cells (mammalian cells). • The acquisition of the Rv0986-8 operon may have increased the fitness of the M. tuberculosis population, being therefore subject to positive selection

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