| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene mutation
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27353755
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| 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 |
see below
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| Other information parent line | The parent parasite line is an UIS4/FLP deleter clone (RMgm-748?). that expresses the yeast Flpl recombinase under the control of the uis4 promoter. The mutant does not contain a drug-selectable marker |
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| The mutant parasite was generated by |
| Name PI/Researcher | Voss C; Sinnis P; Coppens I |
| Name Group/Department | Department of Molecular Microbiology and Immunology, Malaria Research Institute |
| Name Institute | Johns Hopkins University Bloomberg School of Public Health |
| City | Baltimore, Maryland |
| Country | USA |
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| Name of the mutant parasite |
| RMgm number | RMgm-1476 |
| Principal name | ATG8-FRT |
| Alternative name | PbATG8-OE |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not different from wild type |
| Oocyst | Not different from wild type |
| Sporozoite | Not different from wild type |
| Liver stage | ATG8-FRT sporozoites are unable to initiate a blood-stage infection. ATG8-FRT parasites poorly develop into late liver stages. At the onset of infection and until 21 h p.i., the mutant population developed normally, without any significant difference in PV morphology or size from parasites from the parental strain. However, ATG8-FRT parasites significantly suffered a mid-liver-stage developmental defect, more evident from 40 h p.i.
ATG8-FRT parasites produce small merosomes with poorly differentiated merozoites. |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of atg8. The 3'UTR of the atg8 gene is mutated resulting in over-expression of ATG8. The mutation is induced by introducing a construct that was aimed at 'conditional knock-out' of atg8 using the Flp/FRT site-specific recombination system. Introduction of the construct and subsequent removal of the FRT sequence resulted in a mutated 3'UTR of atg8. This mutation did not result in 'knock-out' of atg8 but resulted in overexpression of ATG8
Protein (function)
Autophagy is the archetypal disposal pathway for keeping the cell interior clean of disused and superfluous organelles In contrast to mammalian cells and yeast that contain ~40 autophagy-related genes (ATG), the malaria parasite contains ~15 orthologs of ATG identified by in silico comparative studies: those whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. All the components of the ubiquitin-like ATG8 system, which are involved in autophagosome formation, are expressed by Plasmodium. Coexpression of PbATG8, PbATG3, and PbATG7 is upregulated during early sporozoite differentiation in liver cells. In liver forms of P. berghei, PbATG8 localizes to a network of vesicles and tubules that align along the apicoplast, a relict plastid organelle, as well as to numerous vesicles in the cytoplasm.
PbATG8 is preferentially distributed on the outermost membranes of the apicoplast. ATG8 expressed in the related apicomplexan parasite Toxoplasma gondii is also localized to the apicoplast, and it seems to be involved in the maintenance of this organelle and proper segregation between daughter cells during replication.
Phenotype
The mutant over-expressing PbATG8 showed poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. Based on these and other observations it has been proposed that that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.
Additional information
Evidence is presented for over-expression of atg8 in liver stages, both at the transcript- and protein-level.
Evidence is presented that ATG8-FRT parasites are delayed in the process of microneme elimination and that the apicoplast expands abnormally fast.
Ultrastructural examinations of the merosomal structures produced by ATG8-FRT parasites reveal abnormal budding of merozoites
From the paper:
We then generated a stage-conditional mutant of ATG8 using the FLP/FLP recombination target (FRT) site-specific recombination system in yeast (40), adapted for P. berghei. This method involves the insertion of FRT sites on either side of a genetic element, targeting this region for excision upon expression of the flippase recombinase enzyme (FLP). Following replacement of the endogenous copy of ATG8 with flippase recognition site (FRT)-flanked ATG8 introduced into the UIS4/FLP(-) clone of P. berghei, expression of the FLP occurs under the control of the UIS4 promoter, which is specific for the salivary gland sporozoite stage. This allows normal expression of PbATG8 in blood stages but FLP/FRT-mediated excision that results in silencing PbATG8 function when sporozoites reach the salivary glands.
To bypass the lethality problem of blood forms lacking ATG8, we created stage conditional knockout (cKO) sporozoites in which the ATG8 gene is preserved with its expression driven by the TRAP 3'UTR in blood-stage parasites and the original 3'UTR of the ATG8 gene is interrupted by the plasmid backbone. Upon excision of the TRAP 3'UTR in mosquito salivary glands, the plasmid backbone separates the ATG8 open reading frame (ORF) from its 3'UTR and ensures abrogation of its function. In our cloning strategy, another FRT site was incorporated between the plasmid backbone and the ATG8 3'UTR. Upon excision, the plasmid backbone was removed, allowing the restoration of the ATG8 3'UTR in the UIS4/FLP(-) clone of P. berghei but with an extra 60-bp sequence inserted between the ATG8 ORF and its 3'UTR, as determined by sequencing and verified by PCR
Other mutants
RMgm-1375: Unsuccessful attempts to knock-out atg8
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*RMgm-1476
