Fig 1.
Fitness of promastigote mutants in vitro.
(A) Overview of the experiment timeline for in vitro growth of promastigotes in culture. DNA sampling time-points are indicated by yellow arrows. Dark pink arrows denote dilutions, to (a) 1 x 106 parasites/ml and (b) 1 x 105 parasites/ml. (B) Growth profile of the masterpool of promastigote mutants over time. Data points are the average of three measurements; yellow dots indicate where gDNA was sampled. The dotted line indicates dilution of the cultures. (C) Trajectories of the average of normalised reads of the promastigote masterpool, relative to time-point “0 hours” (T0). Red dotted line highlight relative barcode abundance of 1. Controls are shown in colour: dark blue, SBL1-5 parental cell lines; Cyan, ΔLPG1; Magenta, ΔIFT88; Yellow, ΔPF16. Grey, all other barcoded cell lines. (D) Volcano plot showing fitness scores against p-values of mutants from the promastigote masterpool after 144 hours of growth. Dashed lines demarcate fitness score thresholds of < 0.5 and > 2, and a significance threshold of p < 0.05. Barcodes meeting both threshold criteria are coloured black, non-significant values are grey. Controls are coloured as in D.
Fig 2.
Fitness of promastigote mutants in vivo.
(A) Overview of the experiment timeline for sand fly infections. DNA sampling time-points are indicated by yellow arrows. (B) Fitness scores from the promastigote masterpool after 144 hours in vitro growth plotted against the fitness scores from all mutants 216 hours after infection of sand flies. Black dashed lines mark fitness score thresholds of < 0.5 and > 2. (C-F) Volcano plots showing fitness scores against p-values after 216 h (9 days PBM) in sand flies, separated by sub-pools (P1-P4). Dashed lines demarcate fitness score thresholds of < 0.5 and > 2, and a significance threshold of p < 0.05. Barcodes meeting both threshold criteria are colored black, non-significant are grey. Controls are shown in colour: Dark blue dots, SBL1-5 parental cell lines; Cyan, ΔLPG1; Magenta, ΔIFT88; Yellow, ΔPF16. Black numbers denote the abbreviated GeneIDs (LmxM.xx.xxxx) of selected mutants with the highest fitness changes.
Fig 3.
Pool bottlenecks and required number of flies.
(A) Pool evenness. A small value indicates that the pool is dominated by a few barcoded cell lines with high cell counts. Conversely, a large value means that all cell lines have comparable cell counts. Normal values for pools of cell lines fall into the range 0.7-1.3. (B) Inferred per-fly population bottleneck sizes. Each marker shows the average number of cells that successfully established a detectable infection within a single sand fly for a given replicate, time point and pool according to our mathematical model. The dashed line shows the median population bottleneck size (50 cells per fly). (C) Number of flies used in each pool replicate vs. number of flies required to meet a 90% survival threshold. The solid black line shows the threshold number of flies needed as a function of the pool evenness such that 90% of cell lines survive on average if all cell lines share the same fitness. If this criterion is not met (grey shaded area), it becomes difficult to interpret low-proportion barcodes and extinction as indicators for fitness rather than random events. All pools except for the masterpool lie above the threshold, hence the masterpool was excluded from further analysis.
Fig 4.
V-ATPase is required for differentiation and colonisation in the sand fly vector.
(A) Trajectories of the average of normalised reads for V-ATPase deletion mutants from the promastigote masterpool (grey lines), relative to T0. Dark blue lines, SBL1-5 parental controls. (B) Barcode trajectories for V-ATPase deletion mutants in sand flies, normalised to the start of the experiment. Colour code as in A. (C) Schematic of V-ATPase pump with each subunit labelled. Subunit E, for which the null mutant was individually characterised, is highlighted in magenta. (D) Growth of parental (PAR, dark blue), V-ATPase V1E null mutant (KO, magenta) and V-ATPase V1E add-back (AB, grey) mutant promastigotes in vitro. After 3 days of continuous growth, cultures were diluted back to 1 x 105 parasites/ml and monitored for an additional 3 days. (E) Parasite abundance in the digestive tract of dissected sand flies infected with PAR, AB or KO parasite lines, assessed at 2 days PBM (left) and 9 days PBM (right). (F) Location of PAR, AB or KO parasite lines at 2 days PBM (left) and 9 days PBM (right). NI, non-infected; ES, endoperitrophic space; AMG, abdominal midgut; TMG, thoracic midgut; CAR, cardia; SV, stomodeal valve. (G) Percentage of promastigote morphotypes of PAR, AB or KO parasite lines observed in infected gut smears after 9 days PBM. (H) Sherp transcript quantification by qPCR for PAR, AB and KO parasite lines grown in vitro. Each point shows the abundance of sherp normalised to 18S rRNA, calculated from three replicate measurements. (I) Representative micrographs of promastigote morphotypes found in each cell line in vivo at 9 days PBM. Scale bar = 5 µm.