Proceedings of the 10th International Colloquium on Paratuberculosis

#71 In-Silico identification <strong>of</strong> peptides for <strong>the</strong> diagnostics <strong>of</strong> paratuberculosis
Sheila Tang, Ole Lund, Gregers Jungersen, Morten Nielsen, Heidi Mikkelsen, Claus Lundegaard, Center for
Biological Sequence Analysis, Department <strong>of</strong> Systems Biology, Technical University <strong>of</strong> Denmark, Denmark; National
Veterinary Institute, Technical University <strong>of</strong> Denmark, Denmark
Identification <strong>of</strong> bovine MHC class II reactive peptides that are specific/unique to paratuberculosis and conserved
across pathogenic variations <strong>of</strong> <strong>the</strong> paratuberculosis proteome will be <strong>of</strong> high value for development
<strong>of</strong> new vaccines and immune based diagnostics. Here, we present an in silico screening procedure that can
identify such peptides.
In short, <strong>the</strong> procedure identifies 20mer peptides that fulfill <strong>the</strong> following criteria:
a) Conserved in a set <strong>of</strong> positive (i.e. pathogenic) genomes;
b) Absent in a set <strong>of</strong> negative genomes (i.e. non-pathogenic or related pathogen);
c) High density <strong>of</strong> Bovine MHC-II epitopes.
As positive genomes were used full genome sequences <strong>of</strong> two Mycobacterium avium subsp. paratuberculosis
strains, strain K-10 and <strong>the</strong> newly sequenced strain Ejlskov2007. As negative genomes were used five
complete genomes <strong>of</strong> Mycobacterium tuberculosis, bovis and avium.
All reading frames were virtually translated and converted into overlapping sub-peptide <strong>of</strong> length 20.
Peptides fully conserved in <strong>the</strong> positive strains were selected as potential positive hits. All positive hits were
compared to <strong>the</strong> set <strong>of</strong> 20mers peptides from <strong>the</strong> negative strains and discarded if an overlap <strong>of</strong> 8 consecutive
amino acids was found. This resulted in approximately 80000 20mers that were 100% conserved between <strong>the</strong>
two positive strains, sharing no 8mer overlap to any negative genome.
Residual 20mers were next in-silico checked for binding to each <strong>of</strong> five prevalent bovine class-II MHC
molecules using <strong>the</strong> NetMHCII-pan method [2]. Studies covering Bovine ligands from public data-resources
revealed that this method, even though trained so
References: 1. Larsen, T.S. and A. Krogh, EasyGene-a prokaryotic gene finder that ranks ORFs by statistical
significance. BMC Bioinformatics, 2003. 4: p. 21. 2. Nielsen, M., et al., Quantitative predictions <strong>of</strong> peptide binding
to any HLA-DR molecule <strong>of</strong> known sequence: NetMHCIIpan. PLoS Comput Biol, 2008. 4(7): p. e1000107.
#90 Differential proteome analysis (in vitro vs. in vivo) <strong>of</strong> Mycobacterium avium ssp.
paratuberculosis
Mathias Weigoldt, Klaus Doll, Gerald Friedrich Gerlach, Institute for Microbiology, Department <strong>of</strong> Infectious
Diseases, University <strong>of</strong> Veterinary Medicine Hannover, Hannover, Germany; Clinic for Ruminants and Pigs (Internal
Medicine and Surgery), Justus-Liebig-University, Giessen, Germany
In order to identify novel proteins <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (MAP) expressed in
vivo, MAP was isolated from <strong>the</strong> mucosa <strong>of</strong> four cows with clinical Johne’s disease, fractionated into cytoplasma-
and membrane fractions and compared to <strong>the</strong> respective isolates cultured on Middlebrook Medium 7H9
supplemented with OADC and Mycobactin J. The yield <strong>of</strong> MAP isolated from mucosa was calculated in relation
to <strong>the</strong> mucosa mass and varied from 1.4 % to 4.4 % in three different cows; in one cow <strong>the</strong> yield was below 0.5
% and, <strong>the</strong>refore, not sufficient to study <strong>the</strong> proteome expressed in vivo.
The MAP fractions were subjected to an in-gel trypsin digest and subsequently investigated by UPLC-
Q-ToF-MS/MS analysis using a nano Acquity (Waters) and ESI Q-TOF MS (Q-TOF Ultima, Waters). For <strong>the</strong>
membrane fraction <strong>the</strong> approach resulted in <strong>the</strong> detection <strong>of</strong> more than hundred MAP-proteins expressed in
vivo, and a similar number was detected in vitro. A total <strong>of</strong> 32 different proteins were found to be expressed
in vivo only; five <strong>of</strong> <strong>the</strong>se were expressed in vivo in two cows, and one protein (FO synthase FbiC) was
expressed in vivo only in all three cows. Only one <strong>of</strong> <strong>the</strong> proteins (30S ribosomal protein S2 RpsB) had been
identified in a previous in vivo-in vitro proteome comparison (Stevenson et al., Microbiology 2007; 153, 196-
205). Among <strong>the</strong> o<strong>the</strong>r proteins identified were a number <strong>of</strong> hypo<strong>the</strong>tical proteins <strong>of</strong> unknown function, a
hypo<strong>the</strong>tical fatty acyl dehydrogenase (FadE3_2), and 3-hydroxyacyl-CoA dehydrogenase, all <strong>of</strong> which are
possibly relevant for in vivo-metabolism.
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