Summary

RMgm-4594
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0823300; Gene model (P.falciparum): PF3D7_0922400; Gene product: para-aminobenzoic acid synthetase (aminodeoxychorismate synthase, ADCS)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_0823300; Gene product: para-aminobenzoic acid synthetase (aminodeoxychorismate synthase, ADCS)
Phenotype Asexual bloodstage;
Last modified: 15 March 2019, 14:34
  *RMgm-4594
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30625318
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherMatz JM, Matuschewski K
Name Group/DepartmentDepartment of Molecular Parasitology, Institute of Biology
Name InstituteHumboldt University
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4594
Principal nameacds-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGrowth of asexual blood stages is normal in the presence of pABA in diet of mice. In milk-fed mice adcs- parasite propagation was severely impaired, whereas WT parasites displayed normal growth. Upon pABA supplementation via the drinking water, adcs- growth was fully restored, and mutant parasites grew indistinguishable from WT in milk-fed mice.
Continued pABA restriction led to the retraction of adcs- parasitemia from day 12 onward, which was immediately reverted upon addition of pABA on day 18. During more extended periods of pABA deficiency, adcs- parasitemia dropped below the detection threshold, followed by alternating periods of re-emergence and retraction, indicative of latent malaria infection.
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of ACDS and expresses GFP under the constitutive hsp70 promoter. In addition the selectable marker cassette has been removed by negative selection (see below).

Protein (function)
Malaria parasites replicate at high rates in the mammalian bloodstream. Consequently, they require large quantities of folate and folate precursors, which they scavenge from the serum or synthesize de novo. In contrast, the mammalian host strictly depends on the dietary uptake of preformed folates. Folates promote essential functions during one-carbon transfer reactions and consist of three major moieties: para-aminobenzoate (pABA), pterin, and glutamate.It is believed that chorismate, which is produced via the shikimate pathway, is converted to pABA in a two-step reaction. A potential aminodeoxychorismate synthase (ADCS, PBANKA_0823300) and an aminodeoxychorismate lyase (ADCL, PBANKA_1322100) are predicted by Plasmodium genome sequences.
From the paper: 'We show that the parasite depends on de novo folate synthesis only when dietary intake of pABA by the mammalian host is restricted and that only pABA, rather than fully formed folate, is taken up efficiently. This adaptation, which readily adjusts infection to highly variable pABA levels in the mammalian diet, is specific to blood stages and may have evolved to avoid folate competition between the parasite and its host.'

Phenotype
Growth of asexual blood stages is normal in the presence of pABA in diet of mice. In milk-fed mice adcs- parasite propagation was severely impaired, whereas WT parasites displayed normal growth. Upon pABA supplementation via the drinking water, adcs- growth was fully restored, and mutant parasites grew indistinguishable from WT in milk-fed mice.
Continued pABA restriction led to the retraction of adcs- parasitemia from day 12 onward, which was immediately reverted upon addition of pABA on day 18. During more extended periods of pABA deficiency, adcs- parasitemia dropped below the detection threshold, followed by alternating periods of re-emergence and retraction, indicative of latent malaria infection.

Additional information
Transgenic parasite lines expressing the endogenous acds and adcl genes (RMgm-4596, RMgm-4597) fused to mCherry-3xMyc were generated and analyzed by live fluorescence microscopy. Co-localization with a cytosolic GFP marker revealed that both enzymes localize to the parasite cytoplasm. ADCS expression was restricted to blood stage trophozoites and liver stages, but remained below the detection limit in rings, in schizonts, and throughout mosquito infection. This finding indicates distinct roles for ADCS in the mammalian host only. In contrast, ADCL was ubiquitously expressed during life cycle progression.

'We also compared blood infection of transgenic parasites lacking the second pABA synthesis enzyme, ADCL, in mice receiving different diets. adcl- parasites (RMgm-4595) also depend on nutritional pABA, demonstrating that both enzymes, ADCS and ADCL, promote pABA biogenesis during blood infection. The ubiquitous expression of ADCL suggests additional functions, especially during mosquito stages. A previous study demonstrated a substantial secondary aminotransferase activity of Plasmodium falciparum ADCL, which was suggested as contributing to the detoxification of exogenous D-amino acids'

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0823300
Gene Model P. falciparum ortholog PF3D7_0922400
Gene productpara-aminobenzoic acid synthetase
Gene product: Alternative nameaminodeoxychorismate synthase, ADCS
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationConstructs for endogenous tagging of ADCS and ADCL with mCherry-3xMyc were designed for single homologous integration. The sequences directly upstream of the stop codons were amplified from genomic DNA (1,053 and 1,663 bp, respectively)
and inserted into the pBAT vector using HpaI in combination with EcoRI or NgoMIV. For the generation of the ADCS deletion construct, the 30 fragment of the gene was amplified from genomic DNA (1,185 bp) and cloned into the pBAT vector using the XhoI and KpnI restriction sites. Subsequently, the 50 fragment (508 bp) was amplified and cloned into the intermediate vectors using SacII in combination with HpaI. Prior to transfection, the ADCS knockout vector was linearized with SalI. Deletion of ADCL was performed with the respective NotI-digested PlasmoGEM vector. Internal restriction sites were used for linearization of the tagging vectors.
Additional remarks selection procedureThe adcs– parasites were also subjected to in vivo negative selection with 5-Fluorocytosine-containing drinking water (1 g/l; Sigma-Aldrich, St. Louis, MO, USA) and to subsequent cloning by limiting dilution. Loss of the drug-selectable cassette was confirmed by diagnostic PCR
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationConstructs for endogenous tagging of ADCS and ADCL with mCherry-3xMyc were designed for single homologous integration. The sequences directly upstream of the stop codons were amplified from genomic DNA (1,053 and 1,663 bp, respectively)
and inserted into the pBAT vector using HpaI in combination with EcoRI or NgoMIV. For the generation of the ADCS deletion construct, the 30 fragment of the gene was amplified from genomic DNA (1,185 bp) and cloned into the pBAT vector using the XhoI and KpnI restriction sites. Subsequently, the 50 fragment (508 bp) was amplified and cloned into the intermediate vectors using SacII in combination with HpaI. Prior to transfection, the ADCS knockout vector was linearized with SalI. Deletion of ADCL was performed with the respective NotI-digested PlasmoGEM vector. Internal restriction sites were used for linearization of the tagging vectors.
Additional remarks selection procedureThe adcs– parasites were also subjected to in vivo negative selection with 5-Fluorocytosine-containing drinking water (1 g/l; Sigma-Aldrich, St. Louis, MO, USA) and to subsequent cloning by limiting dilution. Loss of the drug-selectable cassette was confirmed by diagnostic PCR
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0823300
Gene productpara-aminobenzoic acid synthetase
Gene product: Alternative nameaminodeoxychorismate synthase, ADCS
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4