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Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398

The complete genome sequence of Mycobacterium avium subspecies paratuberculosis 27644 Comparative Microbial Genomics. Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398. Outline. Introduction Characteristics of the Map genome Virulence factors and diagnostics

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Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398

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  1. The complete genome sequence of Mycobacteriumavium subspecies paratuberculosis27644 Comparative Microbial Genomics Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398

  2. Outline • Introduction • Characteristics of the Map genome • Virulence factors and diagnostics • Summary

  3. Aug. 2005

  4. Mycobacterium avium subspecies paratuberculosis (Map) • Taxgroup Actinobacteria • Rod shaped • Gram positive, but are hard to stain because of very high lipid content of the cell wall • Resistant to both chemical and environmental changes • High G-C content • Extremely slow growing • Can not produce mycobactin

  5. Johne’s disease • Chronic inflammation of the intestine • Infect all animals especially live stock • Costs 1,5 billion dollars per year in the USA • Three stages – in 2. and 3. bacteria are shed in feces and the animal eventually dies • Contaminates through feces and oral • Difficult to treat because of the high lipid content and complexity of the cell wall; can survive within macrophages • Treatment long and expensive – not feasible in livestock • Has been found in humans with Crohn’s disease

  6. Characteristics of the Map genome • 4,829,781 base pairs • 1 rRNA operon • 4,350 ORFs • 91.30 % codes for proteins • G-C content of 69.3 % • Relatively constant G-C content

  7. Homologous Proteins • 60 % of putative proteins has homologs to other microbial proteins with known functions. • 25 % were homologs to hypothetical proteins • 39 predicted genes are unique to Map • no identical homologous in current databases

  8. Repeats • 1.5 % of Map DNA is repeats (72,7 kb) • Insertion Sequences (IS) • Duplicated householding genes

  9. Insertion Sequences (IS) • 17 copies of IS900 - hypothetical protein • IS_MAP02 – 6 copies – 28 % identity with a transporase in Legionella pneumophila • Some IS are homologous with IS in Mtb, Mav, M. bovis and M. marinum., but there are also IS with no identifiable homologs in other mycobacteria. • Insertion Sequences with no homology in other mycobacteria is of interest, due to their possible usage as diagnostic targets.

  10. Mycobactin production • Map cannot produce Mycobactin in laboratory cultures • Mycobactin is an extracelluary molecule that binds very tightly to iron and transports it into cells.

  11. Mycobactin production • Mtb has a cluster of 10 genes, (mbtA-J),which is responsible for mycobactin production has been found. • Map has a homolog, but it is different in structure

  12. Mycobactin production

  13. Mycobactin production 400 b 551 b 565 b

  14. The virulence of M. avium ssp. paratuberculosis

  15. The virulence of Map Factors that are assumed to be important for the virulence of Map: • The PE/PPE protein family • Membrane lipids • The mce (Mammalian Cell Entry) gene

  16. The PE/PPE protein family • PE/PPE genes comprise 10% of the Mtb genome. In Map, it is only 1 %. • Theory: PE/PPE proteins are there to create antigenic variation

  17. Effects of membrane lipids • Mycobacteria have a lipid-rich, hydrophobic membrane. • This helps the bacteria to survive in a macrophage • Phagosomes containing large hydrophobic granules have been shown to not merge with lysosomes

  18. How to diagnose an M. avium ssp. Paratuberculosis?

  19. Old method: IS900 • Previously, Map was diagnosed using a PCR method, where primers specific to the insertion sequence IS900 were used • But this method was shown to give false positives, as it also detected some other mycobacteria

  20. Unique sequences • 161 unique sequences have been found in Map • A method has been developed, in which PCR is used to target a unique sequence

  21. Summary • Map is the causing agent of Johne’s disease, which costs USD$1.5 billion per year in the USA • Map is unable to produce mycobactin due to a smaller mbtA gene • PE/PPE-proteins, the mce gene and membrane lipids all contribute to the virulence of Map • Unique sequences found in Map can be used to more easily diagnose the bacteria • Parts of the article indicates sloppy work from the authors

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