Proceedings of the 10th International Colloquium on Paratuberculosis

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Structural characterization <strong>of</strong> lipid 550/760 from Mycobacterium avium subspecies
paratuberculosis
Danielle R Cogswell, Donald L Dick, Christopher D Rithner, Julia M Inamine, Torsten M Eckstein
Colorado State University, USA
Mycobacterium avium subspecies paratuberculosis (MAP) is <strong>the</strong> causative agent <strong>of</strong> Johne’s disease which
is a chronic inflammatory bowel disease found in cattle and o<strong>the</strong>r ruminants. Intriguingly, mycobacteria’s cell
envelope is composed <strong>of</strong> more than 40% lipids, most <strong>of</strong> which are found in <strong>the</strong> outer layer <strong>of</strong> <strong>the</strong> cell wall.
This led to a recent research focus to determine <strong>the</strong> role <strong>of</strong> cell wall lipids and <strong>the</strong>ir interactions with host cells.
Recently, it was demonstrated that MAP undergoes a striking change in its lipidome when grown intracellularly
in bovine macrophages. Several lipids <strong>of</strong> MAP that were not seen in-vitro could now be detected which suggested
that <strong>the</strong>y are ei<strong>the</strong>r newly syn<strong>the</strong>sized or over-expressed. It is hypo<strong>the</strong>sized that MAP changes its lipidome
to enhance <strong>the</strong> pathogen’s survival by increasing virulence or by camouflaging <strong>the</strong> surface <strong>of</strong> MAP from
host immune surveillance. One <strong>of</strong> <strong>the</strong>se lipids that is over-expressed under intracellular conditions is lipid spot
550/760. This lipid was also found in a recombinant MAP strain over-expressing MAP0486, a luxR homolog
transcriptional regulator that seems to be responsible for <strong>the</strong> intracellular lipidomic changes. The goal <strong>of</strong> this
research project was to structurally characterize <strong>the</strong> lipid(s) within spot 550/760. Mass spectrometry, 1H-NMR,
1H{13C}-NMR, and 1H1H-TOCSY demonstrated clearly that <strong>the</strong> spot consists <strong>of</strong> two lipid components: mycolic
acids (a-MA, keto/methoxy-MA) and a lipopeptide. The lipopeptide seems to consist <strong>of</strong> a b-OH fatty acyl chain
linked to ei<strong>the</strong>r a single lysine that forms an internal ring through an amide bond with its carboxyl group, or to
two amino acids. However, <strong>the</strong> non-polar character <strong>of</strong> <strong>the</strong> lipopeptide would ra<strong>the</strong>r suggest <strong>the</strong> single lysine.
Fur<strong>the</strong>r NMR and MS/MS analysis will be performed to confirm <strong>the</strong> final structure <strong>of</strong> <strong>the</strong> lipopeptide.
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