Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Abstract

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.

Author Summary

The interplay among host innate immunity and resistance mechanisms in African trypanosomes has a major impact on the host range of these tsetse-fly transmitted parasites, defining their ability to cause disease in humans. A genome-scale RNAi screen identified a highly restricted set of four genes that sensitise trypanosomes to human serum: those encoding the haptoglobin-haemoglobin receptor, a predicted trans-membrane channel, a lysosomal membrane-protein and the cysteine peptidase inhibitor. An analysis of the cysteine peptidases revealed cathepsin-L as the protease regulated by the inhibitor – and with the capacity to render the parasite resistant to lysis by human serum. These findings emphasise the importance of parasite factors for the delivery and stability of host toxins. They also shed light on the control of proteolysis by parasites and potential unanticipated consequences of therapies that target the parasite proteases.