bioRxiv2023
Diseases caused by Leishmania, and Trypanosoma parasites, such as leishmaniasis and African sleeping sickness, are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate and insect hosts, and > 1 billion years of evolutionarily distance, the cell biology of these trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is highly divergent when compared to the other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique -helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family, and is strikingly similar to the structure of WH2 domain, a small actin-binding motif found in many other cytoskeletal regulators. We demonstrate that the WH2-like motif contributes to actin monomer-binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Surprisingly, unlike other profilins characterized so far, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, as well as the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers the molecular principles by which actin dynamics are regulated by profilin in trypanosomatids. Moreover, the unusual actin-binding mechanism of profilin identified here could be applied for designing inhibitors against pathogenic trypanosomatid parasites. AUTHOR SUMMARYLeishmania and Trypanosoma parasites are a major health problem as they cause various diseases in humans and other vertebrates. Currently, there are no specific drugs to treat the diseases caused by these trypanosomatid parasites. Similar to all other eukaryotes, trypanosomatid parasites have an actin cytoskeleton, which is essential for the viability of parasites. Interestingly, both actin and actin-regulatory machineries of these parasites are highly divergent from the ones of animals, making them possible drug targets to treat diseases caused by these parasites. To uncover how the actin cytoskeleton of trypanosomatid parasites is regulated, we focused on a central actin-binding protein, profilin. Importantly, our experiments revealed that trypanosomatid profilins interact with actin through a different structural mechanism as compared to animal profilins, and have specific effects on the assembly of actin filaments. Our genetic studies demonstrate that these specific features of trypanosomatid profilin are also critical for the proper function on this protein in parasites. Our study provides new insight into the cell biology of trypanosomatid parasites. We also envision that the structural and functional differences between trypanosomatid and human profilins can be applied for developing compounds for selectively neutralizing Leishmania and Trypanosoma parasites.