Most African trypanosomes, including the veterinary species Trypanosoma brucei brucei and T. congolense (causative agents of the wasting disease nagana in cattle), are susceptible to lysis by two trypanolytic factors in human serum – TLF1, a component of high density lipoprotein, and TLF2, an IgM/apolipoprotein-A1 complex; both complexes contain apolipoprotein-L1, which forms pores in the parasite’s lysosomal membrane (Vanhollebeke & Pays, 2010).
In contrast, the human-infective sub-species, T. b. rhodesiense and T. b. gambiense, are able to evade lysis by human serum through a variety of mechanisms. Both express truncated variant surface glycoproteins: SRA and TgsGP, respectively (Xong et al, 1998; Uzureau et al, 2013; Capewell et al, 2013). However, whereas SRA prevents APOL1 accessing the lysosome (Stephens et al, 2011), TgsGP expression only serves to hinder the membrane-APOL1 interaction (Uzureau et al, 2013). Instead, human serum resistance in T. b. gambiense also requires reduced lytic factor uptake through the expression of a mutated version of the parasite’s haptoglobin-haemoglobin receptor (Kieft et al, 2010; DeJesus et al, 2013).
Clearly, human serum serum resistance in African trypanosomes has evolved independently on more than one occasion, requiring both gain and loss-of-function mutations. But, are there other proteins that define the sensitivity of some African trypanosomes to lysis by human serum? Are there other routes to human serum resistance? Using this information, can we identify strategies to render resistant parasites sensitive to attack by human serum TLFs? In work funded by the Medical Research Council and the Wellcome Trust, we set out to define the parasite proteins that make T. b. brucei sensitive to the trypanolytic factors in human serum. Using our bloodstream-form RNAi library, we hoped to identify the full set of TLF eficacy determinants expressed by T. b. brucei.
Given the massive size (>0.5 MDa) and complexity of the lytic factors in human serum, we identified surprisingly few loss-of-function mutants that are able to confer significant human serum resistance onto T. b. brucei. As expected, TbHpHbR was the dominant hit in the RNAi library following selection in human serum. In addition, we identified the lysosomal membrane protein, p67, which is known to be important for proper lysosomal function and has previously been shown to influence sensitivity to human serum (Peck et al, 2008). Finally, we identified two proteins not previously known to influence sensitivity to human serum, ‘inhibitor of cysteine peptidase’ (or ICP) and a putative trans-membrane protein (Tb927.8.5240). ICP was subsequently, shown to be important in this pocess by Etienne Pays’ group (Uzureau et al, 2013); however, their analysis did not identify the regulated peptidase presumably responsible for lytic factor destruction.
We focussed our efforts on characterising the role of ICP in determining the human serum sensitivity of T. b. brucei. The parasite expresses two cathepsins, which constitute likely targets for regulation by ICP. Perturbations of CATB and/or CATL by chemical or genetic means had no effect on the sensitivity of wild-type cells to human serum, suggesting that ICP is able to modulate its cathepsin-suppressive effect in response to enzyme activity levels. Loss of ICP resulted in reduced sensitivity to human serum, presumably due to increased cathepsin activity against the lytic factor. Repeating the chemical and genetic perturbations of CATB and CATL in the absence of ICP, revealed that only CATL loss has a dramatic effect on the parasite’s susceptibility to lysis by human serum. Thus, we were able to conclude that ICP’s suppression of CATL activity in T. b. brucei contributes to the sensitivity of these parasites to human serum, sugesting that CATL targets the lytic factor for destruction in the lysosome.
CATL expression is essential for growth of bloodstream-form T. b. brucei, and is now recognised as an excellent target for anti-HAT drug development (Steverding et al, 2012). The above data suggests that its inhibition may render human infective sub-species susceptible to human serum lytic factors. Therefore, drugs that target this enzyme may synergise with trypanolytic factors, rendering otherwise resistant trypanosomes, sensitive to human serum. However, it should be noted that CATL may influence the production and processing of SRA and TgsGP in the endocytic networks of T. b. rhodesiense and T. b. gambiense. So, while targeting CATL may synergise with human serum, we may yet see antagonism resulting in human infective sub-species that are more resistant to human serum. Clearly, further work needs to be done to characterise the complex interactions between these factors in T. b. gambiense and T. b. rhodesiense.