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Plasmodium sporozoites have several surface proteins, including the single-pass transmembrane adhesin thrombospondin related anonymous protein (TRAP). TRAP harbors two extracellular domains, the I-domain and the thrombospondin type I repeat, TSR. TRAP I domain in the “closed” conformation is unbound and has low adhesion and no signaling activity. We use Alphafold to predict the Plasmodium TRAP I domain for mutants design. Here is the Alphafold predicted structures of the TRAP I domain.

Additional file 5: Table S1. Primer list

Additional file 1: Figure S1. Generation of tlp(-)/trap(-) parasites via double homologous recombination. The cartoons (A, B) show the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.

Additional file 3: Figure S3. Generation of trap(-)/trep(-) parasites via double homologous recombination. The cartoon (A, B) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. Plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.

Additional file 2: Figure S2. Generation of tlp(-)/trep(-) parasites via double homologous recombination. The cartoon (A, B) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting Agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.

Additional file 4: Figure S4. Generation of tlp(-)/trep(-)/trap(-) parasites via double homologous recombination. The cartoon (A, B, C) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (D) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (E) Summary of primers and amplicon sizes for the genotyping PCR.

Additional file 3: Figure S3. Generation of trap(-)/trep(-) parasites via double homologous recombination. The cartoon (A, B) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. Plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.

Additional file 4: Figure S4. Generation of tlp(-)/trep(-)/trap(-) parasites via double homologous recombination. The cartoon (A, B, C) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (D) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (E) Summary of primers and amplicon sizes for the genotyping PCR.

PLoS pathogens 13(5), e1006412 - (2017). doi:10.1371/journal.ppat.1006412, Profilinis anactinmonomerbindingproteinthatprovidesATP-actinforincorporationintoactinfilaments.In contrastto highereukaryoticcellswiththeirlargefilamentousactinstruc-tures,apicomplexanparasitestypicallycontainonlyshortandhighlydynamicmicrofila-ments.In apicomplexans, profilinappearsto bethemainmonomer-sequesteringprotein.Comparedto classicalprofilins,apicomplexanprofilinscontainanadditionalarm-likeβ-hair-pinmotif,whichweshowhereto becriticallyinvolvedin actinbinding.Throughcomparativeanalysisusingtwoprofilinmutants,werevealthismotifto beimplicatedin glidingmotilityofPlasmodiumbergheisporozoites,therapidlymigratingformsof a rodentmalariaparasitetransmittedbymosquitoes.Forcemeasurements onmigratingsporozoitesandmoleculardynamicssimulationsindicatethattheinteractionbetweenactinandprofilinfine-tunesglid-ingmotility.Ourdatasuggestthatevolutionarypressureto achieveefficienthigh-speedglid-inghasresultedin a uniqueprofilin-actininterfacein theseparasites., Published by PLoS, Lawrence, Kan.

Background: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. Methods: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). Results: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. Conclusions: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.

Additional file 2: Figure S2. Generation of tlp(-)/trep(-) parasites via double homologous recombination. The cartoon (A, B) shows the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting Agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.

Background: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. Methods: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). Results: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. Conclusions: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.

Background: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. Methods: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). Results: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. Conclusions: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.

Additional file 1: Figure S1. Generation of tlp(-)/trap(-) parasites via double homologous recombination. The cartoons (A, B) show the cloning strategy and primers used for genotyping with amplicon sizes of the resulting transgenic line indicated. Primer sequences are listed in table S2. The plasmid contains mCherry and the resistance marker yFCU for negative selection. (C) Resulting agarose gel picture after genotyping with the expected amplicon sizes given below. (D) Summary of primers and amplicon sizes for the genotyping PCR.