1. The complete genome sequence of Mycobacterium avium subspecies paratuberculosis 27644 Comparative Microbial Genomics Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398

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

3. Aug. 2005

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

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

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

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

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

13. Insertion Sequences (IS) 17 copies of IS900 - hypothetical protein 17 copies of IS900 - hypothetical protein IS_MAP02 – 6 copies – 28 % identity with a transporase in Legionella pneumophila 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. 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. Insertion Sequences with no homology in other mycobacteria is of interest, due to their possible usage as diagnostic targets.

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

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

16. Mycobactin production

17. 400 b 551 b 565 b

18. The virulence of M. avium ssp. paratuberculosis

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

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

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

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

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

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

25. Summary Map is the causing agent of Johne’s disease, which costs USD$1.5 billion per year in the USA 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 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 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 Unique sequences found in Map can be used to more easily diagnose the bacteria Parts of the article indicates sloppy work from the authors Parts of the article indicates sloppy work from the authors