Human African trypanosomiasis is a neglected tropical disease caused by the extracellular protozoan parasite Trypanosoma brucei, and targeted for eradication by 2030. The COVID-19 pandemic contributed to the lengthening of the proposed time frame for eliminating human African trypanosomiasis as control programs were interrupted. Armed with extensive antigenic variation and the depletion of the B cell population during an infectious cycle, attempts to develop a vaccine have remained unachievable. With the absence of a vaccine, control of the disease has relied heavily on intensive screening measures and the use of drugs. The chemotherapeutics previously available for disease management were plagued by issues such as toxicity, resistance, and difficulty in administration. The approval of the latest and first oral drug, fexinidazole, is a major chemotherapeutic achievement for the treatment of human African trypanosomiasis in the past few decades. Timely and accurate diagnosis is essential for effective treatment, while poor compliance and resistance remain outstanding challenges. Drug discovery is on-going, and herein we review the recent advances in anti-trypanosomal drug discovery, including novel potential drug targets. The numerous challenges associated with disease eradication will also be addressed.

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

African trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and spread by the tsetse fly in sub-Saharan Africa. The trypanosome relies on heat shock proteins for survival in the insect vector and mammalian host. Heat shock protein 90 (HSP90) plays a crucial role in the stress response at the cellular level. Inhibition of its interactions with chaperones and co-chaperones is being explored as a potential therapeutic target for numerous diseases. This study provides an in silico overview of HSP90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other trypanosomal species, including non-parasitic Bodo saltans and the insect infecting Crithidia fasciculata. A structural analysis of T. brucei HSP90 revealed differences in the orientation of the linker and C-terminal domain in comparison to human HSP90. Phylogenetic analysis displayed the T. brucei HSP90 proteins clustering into three distinct groups based on subcellular localizations, namely, cytosol, mitochondria, and endoplasmic reticulum. Syntenic analysis of cytosolic HSP90 genes revealed that T. b. brucei encoded for 10 tandem copies, while T. b. gambiense encoded for three tandem copies; Leishmania major (L. major) had the highest gene copy number with 17 tandem copies. The updated information on HSP90 from recently published proteomics on T. brucei was examined for different life cycle stages and subcellular localizations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding a total of twelve putative HSP90 genes, while T. b. gambiense encodes five HSP90 genes. Eighteen putative co-chaperones were identified with one notable absence being cell division cycle 37 (Cdc37). These results provide an updated framework on approaching HSP90 and its interactions as drug targets in the African trypanosome.

The etiological agent of the neglected tropical disease African trypanosomiasis, Trypanosoma brucei, possesses an expanded and diverse repertoire of heat shock proteins, which have been implicated in cytoprotection, differentiation, as well as progression and transmission of the disease. Hsp70 plays a crucial role in proteostasis, and inhibition of its interactions with co-chaperones is emerging as a potential therapeutic target for numerous diseases. In light of genome annotations and the release of the genome sequence of the human infective subspecies, an updated and current in silico overview of the Hsp70/J-protein machinery in both T. brucei brucei and T. brucei gambiense was conducted. Functional, structural, and evolutionary analyses of the T. brucei Hsp70 and J-protein families were performed. The Hsp70 and J-proteins from humans and selected kinetoplastid parasites were used to assist in identifying proteins from T. brucei, as well as the prediction of potential Hsp70–J-protein partnerships. The Hsp70 and J-proteins were mined from numerous genome-wide proteomics studies, which included different lifecycle stages and subcellular localisations. In this study, 12 putative Hsp70 proteins and 67 putative J-proteins were identified to be encoded on the genomes of both T. brucei subspecies. Interestingly there are 6 type III J-proteins that possess tetratricopeptide repeat-containing (TPR) motifs. Overall, it is envisioned that the results of this study will provide a future context for studying the biology of the African trypanosome and evaluating Hsp70 and J-protein interactions as potential drug targets.

The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.