Analysis of released peptidases and their role in the transmission biology of African trypanosomes

Abstract

The protozoan parasite, Trypanosoma brucei causes devastating diseases in both humans and animals in sub-Saharan Africa. They live extracellularly and undergo a complex life cycle involving the mammalian host and the tsetse fly vector. Two developmental forms of the parasite exist in the bloodstream of the mammalian host; the long proliferative slender forms and the cell-cycle arrested stumpy forms. The slender forms differentiate into stumpy forms upon reaching a density threshold through a quorum-sensing like mechanism. During a trypanosome infection, the parasites release peptidases into the bloodstream of their mammalian host which accumulates as parasitaemia increases. These released peptidases hydrolyse their respective host protein substrates resulting in the generation of oligopeptides. At peak parasitaemia, these oligopeptides accumulate and trigger the slender forms to differentiate into non-proliferative stumpy forms. This helps regulate the parasitaemia in the host as well as prepare the parasites for development in the tsetse fly vector. Two peptidases whose activities can provoke the quorum-sensing response have been identified previously in our lab. In this study, proteins secreted/released by the parasites in the bloodstream and early during differentiation to the tsetse fly midgut procyclic forms were analysed by detailed mass spectrometry. This identified twelve peptidases, belonging to different classes of peptidases, enriched at these developmental stages of the parasite. Each peptidase was then validated for its release from parasites using individually epitope-tagged cell lines. Systematic ectopic overexpression and gene knockout using CRISPR/Cas9 of each peptidase gene and their analysis in vivo in mice revealed that two of the peptidases, oligopeptidase B and metallocarboxypeptidase 1, significantly contribute to the generation of the trypanosome’s quorum-sensing signal. Further analysis of peptidases enriched in the secretome of parasites differentiating from stumpy forms to procyclic forms in tsetse flies, however, showed that these peptidases may not be involved in establishing infection in the midgut of the flies but may assist proventricular infection. This work analysed for the first time the contribution of a set of released peptidases by T. brucei involved in the important differentiation from proliferative slender forms into the cell-cycle arrested stumpy forms.