Fig. 1: Dynamics of host cell egress and invasion by B. divergens.
From: Babesia divergens host cell egress is mediated by essential and druggable kinases and proteases
a, Percentage of parasites that egress when treated with 8-Br-cGMP. Data are normalized to an RPMI-treated control (0%). b,c, Time-lapse microscopy of B. divergens egress (b) and invasion (c). Panels b and c follow the same parasite egressing from one cell and invading a new RBC. Coloured arrows indicate the following: red, initial deformation of the RBC in egress; yellow, ‘rounding-up’ associated with permeabilization of the RBC; orange, initial RBC contact during invasion; green, initial deformation of the RBC during invasion; purple, beginning of internalization; blue, completion of invasion; and white, follows the parasite. d, Egress was induced throughout one replication cycle (12 h) of parasites synchronized by merozoite isolation using a range of 8-Br-cGMP concentrations. The half-maximal effective concentration (EC50) of 8-Br-cGMP is shown in teal, and the percentage of parasites that egress at 2 mM 8-Br-cGMP is shown in purple. Data are normalized to 0% being a no-8-Br-cGMP-treated control. e, Percentage of parasites that become motile when the iRBC is permeabilized by saponin, or induced to egress with 8-Br-cGMP, in the stated buffer. IC and EC refer to intracellular (IC, 140 mM K+, 5 mM Na+) and extracellular (EC, 5 mM K+, 140 mM Na+) buffer with or without 2 mM calcium (Ca2+). n is a single cell visualized by live microscopy and is shown for each condition in the x axis. The data are pooled from multiple experiments performed on separate days. f, Screen of small molecules for inhibition of 8-Br-cGMP-mediated induced egress. Data are normalized to 100% using an RPMI-only control and 0% using an RPMI-plus-8-Br-cGMP control. g, B. divergens iRBCs pretreated with cytochalasin D and induced to egress with 8-Br-cGMP. Phalloidin stains the permeabilized RBC. h, Time-lapse images showing RBC permeabilization by cytochalasin D-treated B. divergens during 8-Br-cGMP-induced egress. Phalloidin is excluded from the intact RBC. For b, c and e–h, 500 µM 8-Br-cGMP was used. For a, d and f, each point represents the mean of an individual experiment performed in technical triplicate and n = 3. For a and f, the mean ± s.d. of three biological experiments performed in technical triplicate is shown. Scale bars, 5 µm (b, c and g–h).
