Biology subjects

McNiven, C.

Publications and source records attributed to McNiven, C..

3 recordsLinked to original sources

bioRxiv2026

A pH-dependent protein kinase cascade regulates divergent differentiation of Leishmania in the sand fly.

Leishmania parasites must rapidly adapt to fluctuating environments to ensure survival and transmission. While acidic pH in the sand fly vector is a conserved developmental trigger, sensing mechanisms remain poorly understood. Using a barcoded protein kinase library, we screened for regulators of acid adaptation in Leishmania mexicana, identifying nine protein kinases influencing survival at low pH, including a haptomonad differentiation regulator protein kinase (HDRK1). We demonstrate that HDRK1 null mutants ({Delta}hdrk1) are predisposed to differentiate to haptomonad-like forms at low pH. While {Delta}hdrk1 mutants successfully infect the sand fly midgut, they fail to colonise the stomodeal valve, compromising transmission. Integrated transcriptomic and proteomic analyses revealed that at low pH, {Delta}hdrk1 mutants enter a low energy state reminiscent of AMPK-activated cells. We identified a second protein kinase, HDRK2, establishing a pH-dependent signalling pathway that governs the developmental fate of the parasite, directing differentiation towards either mammalian-infective metacyclic or vector-attached haptomonad stages. Finally, our screen revealed that phosphoinositide balance, regulated by the lipid kinases PI4K and PI4P5K, is vital for acid adaptation, and identified two STE transmembrane kinases as potential pH sensors. Together these findings provide a framework for how Leishmania detects and survives acid stress to coordinate its life cycle.

microbiology↗

bioRxiv2026

Ubiquitinome dynamics and regulation by DUB2 during Leishmania differentiation

Leishmaniasis is caused by Leishmania parasites, which undergo cellular adaptation when transitioning from the insect stage (promastigote) to the mammalian stage (amastigote). While the ubiquitin-proteasome system (UPS) is vital for life cycle progression, global ubiquitination dynamics have remained unmapped. We established a quantitative ubiquitinomics workflow for Leishmania mexicana, identifying over 9,100 ubiquitination sites across 38% of the proteome, revealing thousands of stage-specific regulatory events. Promastigote-enriched sites associate with cell motility, while amastigote-enriched sites link to metabolism and glycosome organization. We identified extensive ubiquitination on UPS components, including the essential virulence factor deubiquitinase 2 (DUB2). Using inducible gene deletion and XL-BioID proximitomics, we identified 111 potential DUB2 substrates. High-confidence substrates include the E2 conjugating enzyme UBC2, which is required for differentiation, and SUMO, a critical regulator of ubiquitin crosstalk. The discovery of UBC2 as a substrate of DUB2 directly links ubiquitination with promastigote to amastigote differentiation. Our findings provide a comprehensive map of the Leishmania ubiquitinome and demonstrate that DUB2 acts as a pleiotropic regulator controlling post-translational modifications of essential proteins involved in life cycle progression.

microbiology↗

bioRxiv2026

CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana

Leishmania mexicana, like other trypanosomatids, possesses a unique kinetochore--the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160-bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.

microbiology↗