ZEB_UniWue @ZEB_UniWue@qoto.org

Author summary Human African trypanosomiasis (HAT) is a lethal neglected tropical disease (NTD) transmitted by the bite of infected tsetse flies. The disease is also known as “sleeping sickness”. During the 20th century it caused enormous suffering in the endemic areas in sub-Saharan Africa. HAT transmission last soared in the late 1990s, triggering a renewed, coordinated and very successful control effort. In this paper, we present achievements towards HAT elimination, with a focus on the WHO road map targets for 2020. In particular, reported cases continue to decline, from over 30,000 cases per year at the turn of the century to 663 cases in 2020. Despite the impact of the COVID-19 pandemic, HAT surveillance was largely sustained, and the network of health facilities able to diagnose and treat the disease further expanded. Looking to the future, the World Health Organization (WHO) set bold new targets for HAT in its 2021–2030 road map for NTDs, namely: the elimination of transmission of gambiense HAT, which occurs in western and central Africa, and the elimination as a public health problem of rhodesiense HAT, which is found in eastern and southern Africa. The strong commitment of national health authorities and the international community will be essential if these goals are to be achieved.

The cytostome-cytopharynx complex (SPC) is an endocytic organelle absent from all human trypanosomatid pathogens save Trypanosoma cruzi. Building upon our previous work identifying the myosin motor MyoF as the first enzymatic component of the T. cruzi SPC, we sought to expand our understanding of this organelle by identifying additional protein machinery which contribute to endocytosis. While deletion of MyoF alone did not fully ablate endocytosis, we found that deletion of both MyoF and the similarly localized MyoC produced an endocytic-null phenotype that was rescued upon complementation. To identify potential regulatory components of this motor complex, we pulled down MyoF and identified an SPC-targeted protein that was conserved across a wide range of protozoan lineages. Deletion of this myosin associated protein (MyAP) alone was sufficient to produce an endocytic-null phenotype, which we were able to fully rescue via complementation. The deletion of MyAP also caused the mis-localization of both cytopharynx myosins to the cytosol. While MyAP lacking the EF-hand domain was unable to complement endocytosis, it was sufficient to restore proper myosin localization. This suggested that MyAP plays two distinct roles, one in targeting myosins to the SPC and a second in regulating myosin motor activity. Lipidomic analysis subsequently revealed a dramatic reduction in the scavenged cholesterol content in the endocytic-null mutants. Overall, this work showcases the first viable endocytic-null mutants generated in T. cruzi through specific gene deletion and highlights the feasibility of leveraging this strategy towards a full dissection of the endocytic machinery and biogenesis of the SPC. ### Competing Interest Statement The authors have declared no competing interest.

Author summary Trypanosoma brucei causes severe diseases that affect humans and livestock in Sub-Saharan Africa. Efficient strategies for manipulating the T. brucei genome have provided a wealth of information about protein localization and function in diverse cellular processes. However, employing live-cell imaging for phenotypic analysis in T. brucei remains a significant challenge because immobilization of this highly motile parasite rapidly leads to morphologic defects and cell death. While fixed-cell imaging can provide snapshots of cellular events, it cannot provide the direct causal link or precise timing of events that comes from watching a living cell change over time. Our strategy using agarose microwells now allows long-term live cell T. brucei imaging with a simple apparatus that can be adapted for a wide range of experimental conditions.