Homology modelling of Trypanosoma brucei major surface proteases and molecular docking of variant surface glycoproteins and inhibitor ligands for drug design.

Trypanosomes, which cause animal African trypanosomiasis, escape host immune responses by renewing their variable surface glycoprotein (VSG) coat. Chemotherapy is currently the only form of external intervention available. However, the efficacy of current trypanocides is poor due to overuse leading to an increase in drug resistance. Major surface proteases (MSPs) of trypanosomes, which are zinc-dependent metalloproteases, are possible drug targets. A Trypanosoma brucei MSP-B (TbMSP-B) mediates parasite antigenic variation via cleavage of 60% of VSG molecules. Whilst TbMSP-A has no apparent role in VSG cleavage; it is not known if TbMSP-C is involved in VSG cleavage. In this study, three-dimensional structures of TbMSP-A, TbMSP-B and TbMSP-C were modelled. By comparing the docking poses of the C-terminal domains of VSG substrates into the models, TbMSP-C showed an affinity for similar VSG substrate sites as TbMSP-B, but these sites differed from those recognised by TbMSP-A. This observation suggests that TbMSP-C may be involved in VSG cleavage during antigenic variation. Furthermore, by docking small inhibitor ligands into the TbMSP-B and TbMSP-C homology models, followed by molecular dynamics simulations, ligands with potential anti-trypanosomal activity were identified. Docking studies also revealed the depth of the S1' pockets of TbMSP-B and TbMSP-C, which is influential in ligand and substrate binding, thereby identifying the protease subsite pocket that should be targeted in drug design.

Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.