The mathematical model for <i>T</i>. <i>brucei</i> infection dynamics.

<p>(A) Schematic of the mathematical model. Slender cells can become committed to differentiation via a SIF dependent route, proportional to SIF concentration, and a SIF independent route. SIF is produced by both committed and non-committed slender forms, and is cleared over time. The concentration of each cell type depends on the replication rate (applicable to slender forms only), the immune clearance rate, the lifespan of that cell type and the differentiation rate (applicable to slender forms only). (B) Standardised residuals (blue circles) of parasite density and slender fraction, by time (dpi, days post infection), of the model fits with SIF-dependent and -independent differentiation to all mice. Under a true model standardised residuals have an approximately standard normal distribution (i.e., zero mean and unit standard deviation (SD)). Inadequate fit of a model is indicated by its residuals deviating from a standard normal distribution (such as residuals further than ~3 SD from zero, represented by the lightest grey shading, or a set of residuals consistently above or below zero. The red line shows the average, across all mice, of the residuals at a particular time point.</p

Comparison of mathematical model and data from mouse infections.

<p>The data is represented by filled dots. The coloured lines represent median fits of the model (including terms for both SIF-dependent and -independent differentiation); the shaded regions indicate 95% predictive intervals, where 95% of future data would be predicted to lie according to the model and the data already observed. (A) Levels of overall parasitaemia over the course of the experiment. (B) Levels of parasitaemia for slender forms only. The complete analysis is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.s003" target="_blank">S3 Fig</a>; dpi, days post infection.</p

Proposed mitochondrial energy metabolism of stumpy form <i>T</i>. <i>brucei</i> cells in the bloodstream.

<p>Schematic representation of key functions we propose to be involved in energy metabolism of stumpy form <i>T</i>. <i>brucei</i>, based on data presented in this work and in earlier studies, as cited in the text. Note that energy metabolism in other compartments such as adipose tissue or skin will very likely be different. Transporters in the inner mitochondrial membrane are shown as coloured squares (MPC, mitochondrial pyruvate carrier; KGC, α-KG carrier; AAC, ATP/ADP carrier). A two-subunit mitochondrial pyruvate carrier, MPC1/2, presumably driven by proton symport, has been identified in <i>T</i>. <i>brucei</i>, but functional studies concluded that at least one additional mitochondrial pyruvate transporter must be present [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.ref074" target="_blank">74</a>], indicated here by a yellow square with a question mark. Enzymes or enzyme complexes associated with the inner membrane are shown as coloured circles (cI, NADH:ubiquinone oxidoreductase; cV, F₁F_O-ATPase, or respiratory complex V; G3P-DH, glycerol-3-phosphate dehydrogenase; AOX, alternative oxidase; NDH2, type 2 NADH dehydrogenase). Functions that directly depend on kDNA-encoded proteins are indicated by red letters and arrows. Key metabolic reactions in the mitochondrial matrix are indicated by numbers in circles: 1, pyruvate dehydrogenase; 2, acetyl-CoA thioesterase; 3, ASCT; 4, SCoAS; 5, α-KG dehydrogenase complex; 6, L-alanine aminotransferase (co-substrate glutamate and co-product alanine omitted for simplicity). Other abbreviations: UQ, ubiquinone; G3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; ACoA, acetyl-CoA; SucCoA, succinyl-CoA).</p

Stumpy forms without kDNA die rapidly when α-KG is the main carbon source.

<p>(A) Stumpy forms of the genotypes indicated were harvested, purified from blood and placed in Creek’s minimal medium (CMM) with 10% (v/v) FCS, supplemented with either glucose (blue bars) or α-KG (grey bars). N-acetyl glucosamine (GlcNAc, 50 mM) was added to one set of experiments to reduce uptake of residual glucose from FCS (red bars). Cells were stained with PI and the % of live cells was assessed by flow cytometry before (t₀; black) and 24 h after the start of the experiment; n = 3 for each cell line; all three kDNA⁰ cell lines were assessed and data averaged (total n = 9). The gating strategy is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.s005" target="_blank">S5B Fig</a>. (B) Quantification of dead cells within WT/WTγ cell populations after 24 h in CMM supplemented with either glucose (25 mM) or α-KG (25 mM), with or without azide (0.5 mM). Cells were stained with PI and analysed by flow cytometry, the gating strategy is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.s005" target="_blank">S5B Fig</a>. Shown are average values ±SD; n = 3.</p

Stumpy form parasites lacking kDNA have a reduced life span <i>in vitro</i>.

<p>(A) <i>T</i>. <i>brucei</i> stumpy forms were harvested from mice, purified from blood and cultured in HMI-9 medium containing 50 μM DFMO to suppress slender form growth. Every 8 h cell numbers were determined in a Coulter particle counter. Cell line WT/L262Pγ (kDNA⁰) #3 had lost its kinetoplast spontaneously, without acriflavine treatment. (B) At every time point shown in panel A, 1x10⁶ cells were stained with CFDA-SE and analysed by flow cytometry to determine the percentage of cells that were dead. The gating strategy is shown in supplementary data. Number of replicates n = 3, with error bars showing SEM.</p

Parasite kDNA is not required for stumpy formation.

<p>(A) Representative phase contrast images showing the morphology of cells at days 4–8 of <i>in vivo</i> infection. Corresponding parasitaemia graphs are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007195#ppat.1007195.g002" target="_blank">Fig 2</a>. Blood smears were taken daily and DAPI-stained, allowing positioning of the nucleus and kDNA to also be assessed. Scale bars represent 10 μm. (B) Immunoblot analysis of expression of the stumpy-specific protein PAD1. Cells were harvested at peak parasitaemia (day 6). 2x10⁶ cells of each cell line were loaded per lane. PCF = procyclic form Lister 427 29.13; SL = slender BSF of parental EATRO 1125 AnTat1.1 90:13 cell line.</p

Average parameter estimates for all cell lines.

<p>Shown are parameter estimates determined from the model with both SIF-dependent and -independent differentiation terms.</p

Stumpy forms lacking kDNA persist for a reduced time span during the first peak of parasitaemia.

<p>(A) Parasitaemia during mouse infections was measured over time. Mice were infected at day 0, with four mice infected per cell line. Error bars are standard error of the mean (SEM). Tail snips were performed every 6 h from day 4 to day 10 post infection and cell counts were estimated from blood smears. Dashed grey line indicates accurate detection limit via the Rapid Matching method. (B) Data from panel A (up to day 6) plotted on a semi-log scale. (C) Fold-change in parasitaemia between time points (up to day 6). (D-H) The morphological changes occurring during these mouse infections, expressed as cell densities (D, slender forms; E, stumpy forms) or percentages (F, slender cells; G, intermediate cells; H, stumpy cells). Error bars are SEM. The population of cells were scored as having slender, intermediate or stumpy form morphology from DAPI-stained dry blood smears. (I) The levels of parasitaemia of stumpy forms per ml of blood, where t = 0 is the time point where the stumpy number was highest for each cell line. This time point was 6 days and 16 hours for cell lines WT/WTγ, WT/L262Pγ #2 and WT/L262Pγ (kDNA⁰) #2, and 7 days and 12 hours for WT/L262Pγ (kDNA⁰) #1. Error bars are SEM.</p

Variables and parameters used in the mathematical model.

<p>Variables and parameters used in the mathematical model.</p

Cell lines used in this study.

<p>Cell lines used in this study.</p