| Malaria,a vector-borne blood disease caused by protozoan parasites of the genus Plasmodium in 91 countries all over the world,leading to an around 400,000 deaths in the year of 2016(WHO,2016).There is no antimalarial vaccine currently in clinical use.Artemisinin combination therapies(ACTs)are the first-line treatment for uncomplicated falciparum malaria in all endemic countries.However,in the Greater Mekong subregion(GMS),artemisinin resistance is increasingly compounded by partner drug resistance,putting ACTs under threat.For its containment,an accelerated development of novel antimalarials will be needed.The malaria parasite are obligate intracellular parasites that relies primarily on their hosts as a nutrient source to fuel its rapid growth and reproduction.They scavenge essential nutrients from their hosts via membrane transport proteins(MTPs).MTPs are molecular targets of most number of FDA-approved drugs,they play important physiological roles in nutrient uptake,disposal of waste products,and shuttling of metabolites between organelles.P.falciparum encodes more than 100 MTPs.In the search for targets for novel prophylactic,therapeutic and transmission-blocking intervention strategies to fight malaria,we report here a broad characterization of the importance of MTPs about folate,amino acids and sugars during the complete life cycle of the murine malaria model parasite Plasmodium yoelii by experimental genetics approaches.We first obtained knockout parasites of different MTP families using CRISPR/Cas9 system,and assessed the function of MTPs in the Plasmodium life cycle.We found that ft1 deletion with no apparent deficiency in life cycle progression,it may be a dispensable gene.FT2 was inherited through the female cell and localized to the crystalloid—a unique organelle of the Plasmodium ookinete.FT2 was important for normal oocyst maturation and sporozoite formation.Moreover,we showed that the number of Aft2 sporozoites in infected mosquitoes was significantly reduced compared to WT in salivary glands,the salivary glands sporozoites displayed slow motility and abrogated transmission from mosquitos to mice by bite-back.We complemented the Aft2 parasite line with Pyft2 and Pfft2,then found that the introduction of a WT copy of Pyft2 or Pfft2 into the genome of Δft2 parasites both rescued salivary gland sporozoites formation.We demonstrated the key role for FT2 during the late stage of oocyst development,which subsequently affects differentiation and sporogony,as well as unveiling a conserved role of FT2 from human and rodent malaria species during development in mosquito.There were five menbers in amino acid transporter family.aat3 was refractory to repeated gene deletion attempts,strongly indicating essential roles during asexual blood-stage growth.aat6 had a function during asexual erythrocytic development,since two independent mutants both showed delayed growth in mice.This screen identified aatl and aat5 deletions with no apparent deficiency at any stage,indicative of dispensable and/or redundant roles for parasite propagation and host switch.aat4 mutants displayed defects during mosquito-stage development.They blocked completely in salivary gland sporozoites formation.AAT4 was essential for sporozoite egress from oocysts,as well as mutants of midgut sporozoites and hemocoel sporozoites were completely compromised in their ability to follow mosquito-to-mouse transition.AAT4 was readily detected in the oocyst periphery at day 7.Peripheral localization was maintained in oocysts before sporozoite formation.When sporozoites had formed and were ready to egress from the oocyst,AAT4 was relocalized to the oocyst cytoplasm.The change of location indicated that AAT4 may perform different functions at different times.AAT4 transported amino acid into oocyst before sporozoite formation,while it facilitated sporozoite egress after oocyst maturation.We were not able to obtain a knockout of st2 in parasites transfected with the knockout vector.This suggested that the st2 gene was essential for survival of asexual stages of P.yoelii.Moreover,we did not observe any phenotype in Plasmodium life cycle for any of the six mutants but st3,suggesting redundancy for each during different development stages.ST3 was important for ookinete to oocyst transformation and oocyst development.We show that deletion of st3 resulted in defects in number,size and growth of oocysts.As a result,none sporozoites were observed in salivary glands.The oocyst stage is the largest bottleneck in the life cycle of the parasite,with the lowest numbers of parasites present in both the mosquito and human host,arguably making this stage the most vulnerable point for disrupting malaria parasite transmission.We used the CRISPR/Cas9 system to produce the largest collection of P.yoelii knock-out parasites available to date.Our data show that MTPs play a prominent role during mosquito-stage development.These results provide genetic support for prioritizing MTPs as targets for novel antimalarials and transmission-blocking vaccines. |
