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) : 27324409 |
MR4 number |
|
top of page |
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 | |
top of page |
The mutant parasite was generated by |
Name PI/Researcher | Shears MJ; McFadden GI |
Name Group/Department | University of Melbourne, BioSciences |
Name Institute | University of Melbourne, BioSciences |
City | Melbourne |
Country | Australia |
top of page |
Name of the mutant parasite |
RMgm number | RMgm-1468 |
Principal name | Pb apiG3PAT(-) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
top of page |
Phenotype |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Reduced infectivity of sporozoites as shown by infection of mice by mosquito bite or injection of purified sporozoites (prolonged prepatent period and/or reduced number of mice developing blood infections) Pb apiG3PAT (-) and wild type sporozoites did not differ in their ability to invade hepatocytes. No difference in mean parasite size was observed between Pb apiG3PAT (-) and wild type parasites at 24 hours post-infection. However, at 48 and 66 hours post-infection, Pb apiG3PAT (-) parasites were significantly smaller than wild type (with mean areas 22 % and 26 % less than the control).
Wild type parasites produced the majority of detached cells and merosomes at 66 hours post-infection, with smaller numbers of these forms detected at the 72 hours. By contrast, Pb apiG3PAT parasites were never seen to produce detached cells or merosomes at either 66 or 72 hours. Pb apiG3PAT (-) parasites had noticeably smaller apicoplasts and fewer nuclei than wild type at 48 and 66 hours. Normal MSP1 expression at 48h. At 66 hours, with MSP1 staining typically detected around individual merozoites in the wild type, but remaining patchy or indicative of only limited plasma membrane invagination in Pb apiG3PAT (-) parasites. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of G3PAT (Pb apiG3PAT)
Protein (function)
Fatty acids are a major component of the phospholipids that make up all cellular membranes and one hypothesis would be that the fatty acids are required for the enormous amount of membrane biosynthesis necessary late in liver stage development for the formation of tens of thousands of exoerythrocytic merozoites. The precursor of phospholipid biosynthesis is phosphatidic acid. Phosphatidic acid is formed in a three-step enzymatic reaction, the first step of which is the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (G3PDH). Two fatty acid side chains are then transferred to glycerol 3-phosphate, firstly by glycerol 3-phosphate acyltransferase (G3PAT) to form lysophosphatidic acid and then lysophosphatidic acid acyltranferase (LPAAT) to form phosphatidic acid. Analysis of the Plasmodium genome has also uncovered two sets of genes for phosphatidic acid biosynthesis and one set is predicted to target to the apicoplast. In the rodent malaria parasite P. yoelii G3PDH and G3PAT are indeed ocalized to the apicoplast only during liver stage development, where they are essential for production of viable merozoites (RMgm-977, RMgm-978).
Phenotype
Reduced infectivity of sporozoites as shown by infection of mice by mosquito bite or injection of purified sporozoites (prolonged prepatent period and/or reduced number of mice developing blood infections) Pb apiG3PAT (-) and wild type sporozoites did not differ in their ability to invade hepatocytes. No difference in mean parasite size was observed between Pb apiG3PAT (-) and wild type parasites at 24 hours post-infection. However, at 48 and 66 hours post-infection, Pb apiG3PAT (-) parasites were significantly smaller than wild type (with mean areas 22 % and 26 % less than the control).
Wild type parasites produced the majority of detached cells and merosomes at 66 hours post-infection, with smaller numbers of these forms detected at the 72 hours. By contrast, Pb apiG3PAT parasites were never seen to produce detached cells or merosomes at either 66 or 72 hours. Pb apiG3PAT (-) parasites had noticeably smaller apicoplasts and fewer nuclei than wild type at 48 and 66 hours. Normal MSP1 expression at 48h. At 66 hours, with MSP1 staining typically detected around individual merozoites in the wild type, but remaining patchy or indicative of only limited plasma membrane invagination in Pb apiG3PAT (-) parasites.
Additional information
Other mutants
RMgm-977, RMgm-978 P. yoelii mutants lacking expressing G3PAT or expressing a Myc-tagged version of G3PAT, respectively.
RMgm-1468: a P. berghei mutant expressing a C-terminal HA-GFP tagged version of G3PAT
|