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

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

Extracellular release of two peptidases dominates generation of the trypanosome quorum-sensing signal

Trypanosomes causing African sleeping sickness use quorum-sensing (QS) to generate transmission-competent stumpy forms in mammalian hosts. This density-dependent process is signalled by oligopeptides that stimulate the signal transduction pathway leading to stumpy formation. Here, using mass spectrometry analysis, we identify peptidases released by trypanosomes and, for 12 peptidases, confirm their extracellular delivery. Thereafter, we determine the contribution of each peptidase to QS signal production using systematic inducible overexpression in vivo, and confirm this activity operates through the physiological QS signalling pathway. Gene knockout of the QS-active peptidases identifies two enzymes, oligopeptidase B and metallocarboxypeptidase 1, that significantly reduce QS when ablated individually. Further, combinatorial gene knockout of both peptidases confirms their dominance in the generation of the QS signal, with peptidase release of oligopeptidase B mediated via an unconventional protein secretion pathway. This work identifies how the QS signal driving trypanosome virulence and transmission is generated in mammalian hosts

In Vitro Assessment of Anthelmintic Activities of Rauwolfia vomitoria (Apocynaceae) Stem Bark and Roots against Parasitic Stages of Schistosoma mansoni and Cytotoxic Study

Schistosomiasis is a Neglected Tropical Diseases which can be prevented with mass deworming chemotherapy. The reliance on a single drug, praziquantel, is a motivation for the search of novel antischistosomal compounds. This study investigated the anthelmintic activity of the stem bark and roots of Rauwolfia vomitoria against two life stages of Schistosoma mansoni. Both plant parts were found to be active against cercariae and adult worms. Within 2 h of exposure all cercariae were killed at a concentration range of 62.5–1000 µg/mL and 250–1000 µg/mL of R. vomitoria stem bark and roots, respectively. The LC50 values determined for the stem bark after 1 and 2 h of exposure were 207.4 and 61.18 µg/mL, respectively. All adult worms exposed to the concentrations range of 250–1000 µg/mL for both plant parts died within 120 h of incubation. The cytotoxic effects against HepG2 and Chang liver cell assessed using MTT assay method indicated that both plant extracts which were inhibitory to the proliferation of cell lines with IC50 > 20 μg/mL appear to be safe. This report provides the first evidence of in vitro schistosomicidal potency of R. vomitoria with the stem bark being moderately, but relatively, more active and selective against schistosome parasites. This suggests the presence of promising medicinal constituent(s)