Figure 5

PBANKA_072090 is translationally repressed in gametocytes, translated in ookinetes and an essential factor for sporozoite development. (A) RT-PCR analysis shows correctly spliced, GFP-tagged mRNA in blood stage gametocytes. p28 serves as control gene. (B) Live imaging of pbanka_072090::gfp parasites shows no expression in gametocytes while GFP (in green) is localized to discrete foci in ookinetes retrieved from a mosquito blood meal. Arrowheads indicate DIC-visible hemozoin clusters. Scale bar = 5 μm. (C) Null mutants parasites show reduced oocyst numbers on days 12 to 13 p.i. Wild type (WT; six independent experiments, n = 88; null mutant five independent experiments, n = 92). All values here (and below) are mean ± SEM; P values were obtained by Mann-Whitney test. (D) Mutant oocysts lack signs of sporulation and appear empty. On the contrary, WT oocysts have already formed sporozoites by day 16 p.i. (arrows). The plasma membrane (pm) of the oocyst is surrounded by a protective capsule. Scale bar = 30 μm. (E) Mutant parasites fail to develop sporozoite (Spz) and colonize salivary glands (SG). Absolute numbers of sporozoites per mosquito from five independent experiments are presented for both WT (n = 6) and null mutant (n = 11) parasites. (F) Mice bitten by mutant-infected mosquitoes do not develop blood stage infections (n = 3). iRBC = infected red blood cells; tRBC = total red blood cells (G) Immunofluorescence assay of oocyst-infected midguts at day 14 p.i. shows strongly reduced CSP expression in mutant parasites; DNA replication is also decreased as detected by Hoechst-33342 staining. ↑ CSP indicates extended exposure of same CSP image to the left. Scale bar = 20 μm. (H) Western blot analysis of oocyst-infected midguts from day 13 p.i. confirms reduced CSP expression in mutants. HSP70 serves as parasite loading control. ↑ CSP indicates extended exposure of same CSP blot above; when CSP can be detected in mutant oocysts, signal from WT oocyst is already saturated.