Identification of a mannose-binding lectin essential for full resistance of Trypanosoma brucei gambiense to the lytic action of the human serum.

Among the three T. brucei subspecies, two (T. b. gambiense and T. b. rhodesiense) are lethal for humans, causing African sleeping sickness, while T. b. brucei is sensitive to the Apolipoprotein 1 (ApoL1), the major compound of the trypanolytic factor (TLF) present in the normal human serum (NHS). T. b. rhodesiense resists lysis by NHS by expressing the Serum Resistant Associated (SRA) protein that directly blocks the action of ApoL1. The resistance mechanism of T. b. gambiense appears more complex, involving multiple factors such as alterations in TLF endocytosis in the required receptor, expression of the T. b. gambiense-specific glycoprotein (TgsGP) and reduction of sensitivity to ApoL1 through altered cysteine protease activity. To discover additional factors involved in the resistance of T. b. gambiense to NHS, a gain-of-function strategy was conducted, consisting in the expression of a T. b. gambiense genomic library in an NHS sensitive T. brucei cell line. After 1% fresh NHS selection, clones able to grow in such conditions were isolated and sequenced to identify the inserted DNA fragment. We identified a gene that confers a 64-fold increase NHS-resistance compared to the T. b. brucei parental cell line when over-expressed. The N-terminal domain of this gene encodes a conserved protein that belongs to the mannose-binding lectin (MBL) family, while the C-terminal domain is specific to T. brucei spp but with unknown function. Involvement of TbMBL in NHS-resistance was further assessed by generating a TbMBL inducible knock-down cell line in T. b. gambiense. A 50% reduction in growth rate was observed when this induced cell line was challenged with 10% NHS. Data of colocalization analysis by immunofluorescence and proximity ligation assay (PLA) not only suggest that TbMBL traffics in the endo/exocytic pathway, but also that TbMBL partially colocalizes with TgsGP. To strengthen this data, co-immunoprecipitation (co-IP) was performed and TbMBL/TgsGP interaction was confirmed. Finally, by proteomic analysis, an interaction of MBL with the Haptoglobin-related protein (HPR), one of the components of the TLF, was revealed. Altogether, our data suggest an essential role of TbMBL in the full resistance of T. b. gambiense against the lytic NHS action