Gene-by-gene screen of the unknown proteins encoded onP. falciparumchromosome 3

Abstract

Taxa-specific proteins are key determinants defining the biology of all organisms and rep-resent prime drug targets in pathogens. How-ever, lacking comparability with proteins in other lineages makes them particularly diffi-cult to study. In malaria parasites this is exac-erbated by technical limitations. Here, we ana-lysed the cellular location, essentiality, func-tion and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nu-cleus was the most common localisation, in-dicating it is a hotspot of parasite-specific bi-ology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The novel mitosis pro-teins defined a possible orphan complex and a highly diverged complex needed for the spin-dle-kinetochore connection. Structure-function comparisons indicated that the taxa-specific proteins evolved by different mecha-nisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biol-ogy of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment inPlasmodiummale gametogenesis

“…We attempted to disrupt the ndc80 gene in the P. yoelii parasite but failed to obtain a mutant clone, suggesting an essential role of Ndc80 in asexual blood stage development. This functional essentiality of Ndc80 is consistent with its expression in asexual blood stage of P. yoelii (Fig S8A) and P. berghei [15], as well as a recent function study of Ndc80 in the P. falciparum schizogony [48]. We used a promoter swap method to replace 801 bp of endogenous ndc80 promoter sequence in the double tagged strain DTS1 with that (1826 bp) of clag1 gene (PY17X_1402200) (Figure 8A), whose transcripts are expressed in asexual stages, but absent in gametocytes and mosquito stages [49].…”

The Kelch13 compartment is a hub of highly divergent vesicle trafficking proteins in malaria parasites

“…Assessing parasite proliferation after knock sideways of KIC12 showed a mean relative growth of 37.0% compared to control parasites after two development cycles, indicating an important function of KIC12 for asexual parasite proliferation (Figure 4D; Figure S5B). Due to the dual localisation of KIC12 we also generated KIC12-2xFKBP-GFP-2xFKBP endo parasites episomally co-expressing an alternative mislocaliser (Lyn-FRB-mCherry) [63], enabling conditional inactivation of the nuclear pool of KIC12-2xFKBP-GFP-2xFKBP by mislocalisation to the parasite plasma membrane (PPM), an approach previously shown to be suitable for efficiently inactivate nuclear proteins [63, 71]. Induction of KIC12 mislocalisation to the PPM resulted in a loss of KIC12 in the nucleus 4 hours post induction (Figure 4E).…”

A microtubule associated protein is essential for malaria parasite transmission

“…Nuclei were stained with 1 µg/mL Hoechst-33342 (Invitrogen). Microtubules were visualized by incubation of parasites in medium containing 1:1,000 TubulinTracker Deep Red (Thermo Fischer Scientific; dissolved in DMSO), which labels polymerized tubulin, for 15 min at 37°C prior to imaging, as previously described (86). Images were processed using Fiji (83) and Adobe Photoshop CC 2021 was used for display purposes only.…”