Evaluation of Clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is anurgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role inpathogenicity and virulence in Leishmania spp. and are promising targets for developing new antileishmanialdrugs. Genetic drug target validation has been performed on a number of cysteinepeptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidasesfor gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertaintheir cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1,LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. Theother peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51,C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant(Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant ofPNT1 (Δpnt1:: PNT1flox/Δ pnt1::HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellumand on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits onthe cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutantPNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival.Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidasesthat are potential drug targets in Leishmania

Divergent cytochrome c maturation system in kinetoplastid protists

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c(1) is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c. KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigen

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface expressed Variant Surface Glycoprotein (VSG), whilst ensuring only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated only in catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signalled to guide the appropriate reaction, or to integrate switching into parasite growth, is unknown. Here we show that loss of ATR, a DNA damage signalling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localised damage. Furthermore, ATR loss leads to increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with altered localisation of RNA Polymerase I and VEX1. This work therefore reveals that ATR acts in antigenic variation both through DNA damage signalling and surface antigen expression control

Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division

CLK1/CLK2 driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics

Kinetochores in the parasite Leishmania and related kinetoplastids appear to be unique amongst eukaryotes and contain protein kinases as core components. Using the kinetochore kinases KKT2, KKT3 and CLK2 as baits, we developed a BirA* proximity biotinylation methodology optimised for sensitivity, XL-BioID, to investigate the composition and function of the Leishmania kinetochore. We could detect many of the predicted components and also discovered two novel kinetochore proteins, KKT24 and KKT26. Using KKT3 tagged with a fast-acting promiscuous biotin ligase variant, we took proximity biotinylation snapshots of the kinetochore in synchronised parasites. To quantify proximal phosphosites at the kinetochore as the parasite progressed through the cell cycle, we further developed a spatially referenced proximity phosphoproteomics approach. This revealed a group of phosphosites at the kinetochore that were highly dynamic during kinetochore assembly. We show that the kinase inhibitor AB1 targets CLK1/CLK2 (KKT10/KKT19) in Leishmania leading to defective cytokinesis. Using AB1 to uncover CLK1/CLK2 driven signalling pathways important for kinetochore function at G2/M, we found a set of 16 inhibitor responsive kinetochore-proximal phosphosites. Our results exploit new proximity labelling approaches to provide a direct analysis of the Leishmania kinetochore, which is emerging as a promising drug target

Chromosomal assembly of the nuclear genome of the endosymbiontbearing trypanosomatid Angomonas deanei

Abstract Angomonas deaneiA. deane