RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5357

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1128300; Gene model (P.falciparum): PF3D7_0629500; Gene product: amino acid transporter AAT1 (AAT1)
Phenotype	Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;

Last modified: 24 May 2023, 16:39

*RMgm-5357

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 36976018
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Anand A, Nagaraj VA
Name Group/Department	Infectious Disease Biology
Name Institute	Institute of Life Sciences
City	Bhubaneswar, Odisha
Country	India
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Name of the mutant parasite
RMgm number	RMgm-5357
Principal name	PbAAT1KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	- A slight growth delay of asexual blood stages in mice - Parasites have swollen food vacuoles (FVs) with less hemozoin - The hemozoin crystals display a thin morphology compared with wild-type parasites. - A significant decrease in the hemozoin (Hz) levels accumulated in organs such as the spleen, liver, lungs, and brain of PbAAT1KO-infected mice - Reduced sensitivity to chloroquine and amodiaquine. - Mice infected with the knockout parasites show reduced neuronal inflammation and cerebral complications
Gametocyte/Gamete	Normal gametocyte production (and sex ratio). Swollen' food vacuoles in male and female gametocytes. In vitro exflagellation analyses performed on infected blood samples showed hardly detectable exflagellation for PbAAT1KO male gametocytes until day 9. However, exflagellation could be detected on day 11, and from day 11 onward, the number of exflagellation centers observed for PbAAT1KO male gametocytes was comparable with PbWT.
Fertilization and ookinete	While there was an almost 94% decrease in the formation of PbAAT1KO ookinetes on day 7, the ookinete numbers were comparable between PbWT and PbAAT1KO on day 12. The dissection of day-7-fed infected mosquito guts 21 h after feeding indicated an almost 90% decrease in ookinetes compared with PbWT. However, feeding mosquitoes with PbAAT1KO-infected mice on day 12 post-infection (p.i.) did not show a significant decrease in the formation of ookinetes. While there was a significant increase in the proportions of PbAAT1KO retorts formed in vitro and in vivo on day 7 p.i., the proportions were comparable on day 12 p.i. between PbWT and PbAAT1KO infections.
Oocyst	The decrease in the number of ookinetes formed in the day-7-fed mosquitoes was further reflected in the number of oocysts observed in the infected mosquito guts dissected on day 10 and the number of sporozoites present in the salivary glands of the mosquitoes dissected on day 19. No such changes were observed in the number of oocysts and sporozoites for day-12-fed PbAAT1KO-infected mosquitoes.
Sporozoite	The decrease in the number of ookinetes formed in the day-7-fed mosquitoes was further reflected in the number of oocysts observed in the infected mosquito guts dissected on day 10 and the number of sporozoites present in the salivary glands of the mosquitoes dissected on day 19. No such changes were observed in the number of oocysts and sporozoites for day-12-fed PbAAT1KO-infected mosquitoes.
Liver stage	No significant differences in the prepatent period and the appearance of asexual-stage parasites in mice intravenously infected with defined numbers of PbWT and PbAAT1KO sporozoites.
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of AAT1. Protein (function) The P. berghei genome encodes a putative AAT1 (PBANKA_1128300) consisting of 614 amino acids. Its orthologue in P. falciparum, PfAAT1 (PF3D7_0629500), has been shown to localize in the food vacuole (FV). It contains 10 to 11 predicted transmembrane domains. Phenotype A slight growth delay of asexual blood stages in mice. Blood stage parasites have swollen food vacuoles (FVs) with less hemozoin (Hz). The Hz crystals display a thin morphology compared with wild-type parasites. A significant decrease in Hz levels accumulated in organs such as the spleen, liver, lungs, and brain of PbAAT1KO-infected mice. Reduced sensitivity to chloroquine and amodiaquine. Mice infected with the knockout parasites show reduced neuronal inflammation and cerebral complications. Normal gametocyte production (and sex ratio). Swollen' food vacuoles in male and female gametocytes. In vitro exflagellation analyses performed on infected blood samples showed hardly detectable exflagellation for PbAAT1KO male gametocytes until day 9. However, exflagellation could be detected on day 11, and from day 11 onward, the number of exflagellation centers observed for PbAAT1KO male gametocytes was comparable with PbWT. While there was an almost 94% decrease in the formation of PbAAT1KO ookinetes on day 7, the ookinete numbers were comparable between PbWT and PbAAT1KO on day 12. The dissection of day-7-fed infected mosquito guts 21 h after feeding indicated an almost 90% decrease in ookinetes compared with PbWT. However, feeding mosquitoes with PbAAT1KO-infected mice on day 12 post-infection (p.i.) did not show a significant decrease in the formation of ookinetes. While there was a significant increase in the proportions of PbAAT1KO retorts formed in vitro and in vivo on day 7 p.i., the proportions were comparable on day 12 p.i. between PbWT and PbAAT1KO infections. The decrease in the number of ookinetes formed in the day-7-fed mosquitoes was further reflected in the number of oocysts observed in the infected mosquito guts dissected on day 10 and the number of sporozoites present in the salivary glands of the mosquitoes dissected on day 19. No such changes were observed in the number of oocysts and sporozoites for day-12-fed PbAAT1KO-infected mosquitoes. No significant differences in the prepatent period and the appearance of asexual-stage parasites in mice intravenously infected with defined numbers of PbWT and PbAAT1KO sporozoites. Additional information Analysis of a mutant expressing an N-terminal GFP-tagged version of AAT1 (RMgm-5356) showed the following: Live fluorescence imaging performed with PbWT(+GFP-AAT1) parasites showed a faint signal probably due to the loss of GFP fluorescence in the acidic environment of food vacuoles (FVs). Indirect immunofluorescence using GFP antibodies showed the colocalization of GFP-AAT1 with discrete FVs containing hemozoin (Hz) in PbWT(+GFP-AAT1) parasites, suggesting the FV localization of PbAAT1. Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1128300

Gene Model P. falciparum ortholog

PF3D7_0629500

Gene product

amino acid transporter AAT1

Gene product: Alternative name

AAT1

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Generation of PbAAT1KO parasites: PbAAT1KO parasites were generated by transfecting PbWT parasites with pL0006 plasmid with 5′ and 3′ UTRs of the AAT1 gene on either side of the human DHFR selectable marker using the double-crossover homologous recombination strategy. PbWT genomic DNA was used to amplify a 699-bp 5′ UTR of PbAAT1 using Phusion DNA polymerase (Thermo) with forward (5′-GCCAGGGCCCTCCATTGCTGCTGTATTTTTATTCTG-3′) and reverse (5′-GCCCAGATCTTCTATTAAAAAATAGATGCAATCATTCATACC-3′) primers. Similarly, a 724-bp 3′ UTR of PbAAT1 was amplified with forward (5′-GCCAGGTACCTGAAAGACCGAAGTGTGCTTTTTACTTTATAC-3′) and reverse (5′-GCCCGCGGCCGCGTGCAGTTTATAAGCCGAGCTTG-3′) primers. The resultant 5′-UTR and 3′-UTR fragments were digested with ApaI and BglII and KpnI and NotI, respectively. Digested fragments were then cloned into pL0006 plasmid flanking the human DHFR expression cassette. The recombinant plasmid was then linearized with ApaI and NotI

Additional remarks selection procedure

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

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