Trypanosome flagellum structure, assembly, and mechanism

University Of California Los Angeles, Los Angeles CA

Investigators

Abstract

PROJECT SUMMARY The flagellated protozoan parasite Trypanosoma brucei is responsible for African trypanosomiasis (i.e., sleeping sickness), which causes widespread mortality and morbidity of humans and livestock in sub-Saharan Africa. Sleeping sickness is fatal if untreated, yet no vaccine exists and diagnostic on the field is limited. T. brucei is also a model organism for other devastating trypanosomatid parasites, notably T. cruzi and Leishmania spp. Therefore, there is a pressing need for research to better understand these parasites and facilitate development of new therapeutic interventions. T. brucei and all trypanosome pathogens depend on their flagellum for motility and signaling in response to the host environment, interaction with host tissues, cell morphogenesis and division. The T. brucei flagellum is demonstrated to be essential for parasite’s transmission through its fly vector and infection of the mammalian host. Significantly, the flagellum of these pathogenic parasites all fashion a prominent, lineage-specific paraflagellar rod (PFR), attached to the conserved axoneme consisting of 9 doublet microtubules (DMT) + 2 central pair microtubules (CP) and projections. By cryogenic electron tomography (cryoET) and microscopy (cryo-EM), we have determined the architecture and molecular arrangement of the intact T. brucei axoneme with PFR and the atomic structures of its isolated DMT with 154 different axonemal proteins, including 40 proteins unique to the trypanosome lineage (Xia et al. Science, in press). These structures and knock-down functional studies point to our overall hypothesis that PFR and other lineage-specific proteins underlie trypanosome-specific motility that drives infection and transmission. We propose to test this hypothesis by determining the entire flagellum structure, mechanisms of assembly, and mechanisms of operation. Specifically, we will employ cutting-edge cryo-EM determine atomic structures of PFR and carry out structure-guided functional and biophysical studies (Aim 1). We will generate models for flagellar assembly and motility based on chemistry of protein side-chain interactions. We then use sophisticated molecular genetics, biophysical and motility analyses to directly interrogate predictions of these models. Aim 2 will focus on the structure and function of T. brucei CP and its associated, lineage-specific projections. Lastly, we will uncover the in situ structures the flagellum as attached to the T. brucei cell (Aim 3). This project harnesses currently the worlds’ most advanced Krios G4 microscope just installed at UCLA, together with mutational analysis and AI-aided data processing methods. The anticipated atomic-resolution models of the flagellum and structure-guided functional studies focused on pathogen-specific proteins will unveil potential therapeutic targets for future exploitation in treating neglected diseases. Broadly, thanks to flagella’s critical role in this and many other pathogenic protozoa, and in normal human development and health, results should be of wide interest for the community studying pathogenesis of parasitic protozoa, human development and physiology, and fundamental biology of eukaryotes.