The shikimate pathway enzyme that generates chorismate is not required for the development of Plasmodium berghei in the mammalian host nor the mosquito vector

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei

“…Failure to knockout SPATR in P. berghei is also consistent with the PlasmoGem results (Bushell et al, ). Hence, the yeast‐based Flp/FRT mutagenesis system was employed to conditionally delete Spatr in the parasite's mosquito stages (Choudhary, Srivastava, Singh, Kumar, & Mishra, ; Combe et al, ; Lacroix et al, ). Flp recognition target (FRT) site was engineered in the only intron present in the gene (Figure b).…”

“…For the generation of p Spatr /FRT targeting construct three fragments F5, F6 and F7 were cloned sequentially into the p3’trap‐hDHFR‐flirte vector (Figure b) and it was transfected in P. berghei ANKA TRAP/FlpL line. Parental TRAP/FlpL parasites express FlpL recombinase under the TRAP promoter, which is active in mosquito stages and selectively excises FRT‐flanked DNA sequence (Choudhary et al, ; Combe et al, ; Lacroix et al, ) S. Mishra, K.A. Kumar, and P. Sinnis, unpublished data).…”

“…Transmission to mosquitoes resulted in successful oocyst formation (Figure a), normal sporulation pattern (Figure b) and similar number of midgut sporozoites (Figure c) in Spatr cKO when compared to TRAP/FlpL parasites. On Day 18, infected mosquito cages were transferred to 25°C for optimal FlpL activity to achieve maximum excision of the flirted locus (Choudhary et al, ). On the next day, similar numbers of Spatr cKO and TRAP/FlpL salivary gland sporozoites were obtained (Figure d) and excision of Spatr locus was evaluated by PCR analysis.…”

“…To elucidate the role of SPATR (PlasmoDB ID: PBANKA_0309500), we used Plasmodium berghei ANKA, a species that infects rodents. The P. berghei ANKA TRAP/FlpL line, which expresses FlpL under the control of the TRAP promoter (Choudhary et al, ; Lacroix et al, ) was transfected to obtain the Spatr cKO parasites. Anopheles stephensi mosquitoes were grown at 28°C and 80% relative humidity.…”

“…Fragments F5 and F7 were subcloned sequentially at Sac II/ Not I and Pst I/ Kpn I sites respectively. Final targeting cassette was separated from vector backbone by digestion with Sac II/ Kpn I and used for transfection into P. berghei ANKA TRAP/FlpL line (Choudhary et al, ; Lacroix et al, ). Transfected parasites were selected by oral administration of pyrimethamine in drinking water.…”