Journal: bioRxiv

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

doi: 10.64898/2026.09.15.751656

Figure Lengend Snippet: A) Bar chart indicating the percentage of population with extra-axonemal material over 7 days in Grace’s media (n >86). Day 0 represents cells before transfer to pH 5.5. Phase-contrast images show examples of cells with the extra-axonemal material. Scale bars, 5 µm. B) Phase-contrast images of mid-log Δhdrkl procyclic promastigotes washed and grown in HOMEM medium at differing pH values, pH 7.4, 6.0 and 5.0, for 24, 48 and 72 h. The percentage of cells with extra-axonemal material indicated in white text. C) Phase-contrast images of Δhdrkl cells grown for 7 days at pH 5.5, then washed and resuspended in HOMEM medium at pH 5.0, 6.0 or 7.4 for 48 h. Scale bars, 5 µm. The percentage of cells with extra-axonemal material is indicated in white text. D) Schematic of the HDRK1 (LmxM.18.0640) protein kinase, with the conserved motifs depicted below. Residues of interest are indicated: residues required for kinase catalytic activity, lysine and aspartic acid, are shown in red; T164, a confirmed phosphosite, is shown in green; and C166, a cysteine, linked to redox activation, is shown in yellow. E) Ribbon diagram of the HDRK1 AlphaFold model with residues of interest highlighted. K38, D132 and D150 are shown in red, T164 in green and C166 in yellow. F) Sequence alignment of HDRK1 and the kinase domain of human AMPKα (AAPK2) in the context of secondary structure elements from the HDRK1 AlphaFold model. G) Bar chart showing the percentage of population with extra-axonemal material for T7Cas9 and HDRK1 precision-edited lines, K38, D132/D150, T164 and C166, grown at low pH for 7 days.

Article Snippet: 10 μg of purified vector was transfected into mid-log phase Leishmania , and cells were selected with 25 μg/mL G418 (InvivoGen) for episomal expression of the reporter.

Techniques: Phase Contrast, Activity Assay, Phospho-proteomics, Activation Assay, Sequencing

Journal: bioRxiv

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

doi: 10.64898/2026.09.15.751656

Figure Lengend Snippet: A) Bar chart showing infection rates and parasite loads for T7Cas9, Δhdrkl and Δhdrk1 /HDRK1 in L. longipalpis at days 1, 4−5 and 9 post blood meal (PBM). Parasite loads were classified as light, <100 parasites per gut; moderate, 100−1,000 parasites per gut; or heavy, >1000 parasites per gut. Data were collected from two experiments, with approximately 10−20 sand fly dissections for each line at each time point. B) Bar chart showing the location of parasites in sand flies at days 1, 4−5 and 9 PBM. The sand fly schematic shows the digestive tract, colour-coded to indicate parasite location: endoperitrophic space (pink); abdominal midgut (AMG; light blue); abdominal and thoracic midgut (AMG/TMG; lilac); abdominal midgut, thoracic midgut and cardia (AMG/TMG/cardia; lime); and colonised stomodeal valve (SV; dark blue). C) Giemsa-stained cells representing the morphological forms found in the day 9 sand fly dissections for T7Cas9, Δhdrkl and Δhdrk1 /HDRK1. Scale bar, 10 pm. D) Bar chart showing the relative representation of individual morphological categories in T7Cas9, Δhdrkl and Δhdrk1 /HDRK1 detected in L. longipalpis at day 9 PBM. E) Image of scratched plastic after incubation with T7Cas9 and Δhdrkl mid-log phase parasites for 24 h, followed by Giemsa staining. Brightfield image acquired at 40x magnification.

Article Snippet: 10 μg of purified vector was transfected into mid-log phase Leishmania , and cells were selected with 25 μg/mL G418 (InvivoGen) for episomal expression of the reporter.

Techniques: Infection, Parasite, Staining, Incubation, Brightfield

Journal: bioRxiv

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

doi: 10.64898/2026.09.15.751656

Figure Lengend Snippet: A) Live-cell image of mNG::HDRK1 in a cell from a mid-log culture grown in HOMEM medium, pH 7.4. The nucleus and kinetoplast shown in magenta. Basal body signal from mNG::HDRK1 is shown in green. Scale bar, 5 µm. B) Localisation of mNG::HDRKl in live cells grown in Grace’s media at pH 7.4 (0) or pH 5.5 for 1-7 days. Scale bar, 5 µm. C) Live-cell imaging in CyGel of mNG::HDRK1 promastigotes attached to a gridded slide. Scale bar, 5 µm. D) Expression levels of myc::HDRK1 over 8 days in low pH (Grace’s media, pH 5.5). OPB was used as a protein loading control. Day 0, mid-log cells in pH 7.4. S, stationary-phase culture in pH 7.4 media. Day 1-8, cells in low pH Grace’s media (pH 5.5). Bar chart showing HDRK1 signal intensity normalised to the OPB control. E) TEM images of Δhdrkl cells grown at low pH for 7 days. Scale bars, 500 nm. F) SEM images of Δhdrkl cells grown at low pH for 7 days. Scale bars, 1 pm. G) Flow cytometry showing the cell cycle stage (G1, S or G2) in T7Cas9 and Δhdrkl cells at pH 7.4 and after 7 days at pH 5.5.

Article Snippet: 10 μg of purified vector was transfected into mid-log phase Leishmania , and cells were selected with 25 μg/mL G418 (InvivoGen) for episomal expression of the reporter.

Techniques: Live Cell Imaging, Expressing, Control, Flow Cytometry

Journal: bioRxiv

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

doi: 10.64898/2026.09.15.751656

Figure Lengend Snippet: A) Phase-contrast images of fixed T7Cas9 and ΔLmxM.36.l520 cells after 7 days in Grace’s media. Scale bar, 10 µm. B) Schematic of HDRK2 (LmxM.36.1520). Canonical protein kinase motifs are shown below the cartoon. HDRK2-specific motifs shown above. The three residues required for kinase activity are highlighted in red. C) Live-cell images showing mNG::HDRK2, green, at days 0, 2 and 4 in low pH, Grace’s media. Hoechst is shown in magenta. D) Live-cell images of Ahdrk2/mNG::HDRK1 at days 0–5 in low pH, Grace’s media. Left, merged image with Hoechst, magenta, and mNeonGreen, green. Right, phase-contrast image. Scale bars, 5 µm. E) Western blot showing HDRK1 expression at 0 (mid-log at pH 7.4), 2, 5 and 8 days in low pH (pH 5.5) in T7Cas9 and Δhdrk2 cells. OPB is shown as a loading control.

Article Snippet: 10 μg of purified vector was transfected into mid-log phase Leishmania , and cells were selected with 25 μg/mL G418 (InvivoGen) for episomal expression of the reporter.

Techniques: Phase Contrast, Activity Assay, Western Blot, Expressing, Control