In spite of a globally similar functional classification, the contribution of proteins involved in signaling and protein synthesis was quite different between the three strains. In addition,

some proteins were specifically identified by one strain (Figure 3) and are therefore potential candidates for strain discrimination and/or to understand their pathogenicity. Other than proteins with no known function, these markers included specific isoforms of adenylate kinase and lysophospholipase in Feo, a dihydrolipoyl dehydrogenase in Biyamina, and a specific isoform of adenine phosphoribosyltransferase and a calpain-like cysteine peptidase, as well as a tryparedoxin for the OK strain. Figure 2 Classification of T. brucei gambiense proteins from 3 different strains into functional categories. Proteins from the different strains (Feo, OK, Biyamina) were classified into 12 functional categories GW-572016 cell line according to the hierarchical, nonredundant classification system developed for MapMan [13]. On the x-axis, the categories are indicated. The y-axis shows the percentage of each category for each strain. Figure 3 Overlap between secretomes of 3 different T. brucei gambiense strains. Proteins found in the analysis of 3 different T. brucei strain secretomes separated on 1D-PAGE were compared. The black circle in the middle represents

proteins common Acalabrutinib ADP ribosylation factor to the 3 strains (48 proteins). Biyamina and OK have 16 proteins in common; 14 proteins are specific to the Biyamina secretome. 2- Secreted proteins form stable complexes To further understand the secretome

and its interaction network, protein complexes were separated using two-dimensional BN-SDS-PAGE (blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis) [14]. With this method, proteins focusing on a virtual vertical lane are potentially part of the same complex, whereas proteins not in a complex are focused at the same molecular weight (MW) in both dimensions and located at the extreme right on the gel (Figure 4). Gels have been carried out two times giving similar protein profiles. A total of 382 nonredundant proteins were identified by MS/MS (additional file 2, Table S2). Functional classification led to a similar distribution as above (see Figure 2). Figure 4 highlights the importance of a small number of protein spots (<20) that accounted for more than 80% of the total amount of secreted proteins. These proteins included not only the well-known and abundant VSGs (spots 33, 182, 43), but also enzymes involved in nucleotide and amino acid metabolism (spots 76, 123, 126), chaperones (spots 114, 113, 89, 107), and proteases (spots 165, 114), thus defining a major role for defense and nutrition to the secretome.