Mutant/mutation
The AMA1^ko mutant  lacks expression of AMA1 and expresses GFP under the control of the constitutive hsp70 promoter. The mutant has not been cloned. Parasite populations have been analysed that consisted of mixtures of the AMA1^ko GFP⁺ mutant parasites and parasites expressing AMA1 (AMA1⁺ GFP^- parasites). Attempts of cloning AMA1^ko GFP+ parasites were unsuccessful.

Protein (function)

Phenotype
The AMA1ko mutant  lacks expression of AMA1 and expresses GFP under the control of the constitutive hsp70 promoter. The mutant has not been cloned. Parasite populations have been analysed that consisted of mixtures of the AMA1ko GFP+ mutant parasites and parasites expressing AMA1 (AMA1+ GFP- parasites).
Evidence is presented that the multiplication rate of AMA1ko GFP+ parasites is 35% that of wild type parasites. The AMA1ko GFP+ schizonts produced normal numbers of meorozoites, indicating that the decreased multiplication rate of AMA1KO parasites reflects a defect in merozoite entry into erythrocytes. Evidence is presented that AMA1 is involved in merozoite attachment to, but not internalization into, the host red blood cell.
To test AMA1KO sporozoite capacity to invade hepatocytes, populations of AMA1ko GFP+/ AMA1+ GFP- parasites were transferred to mosquitoes. The same ratio of GFP+ versus GFP- sporozoites is found in the blood fed to mosquitoes and in the mosquito salivary glands, indicating that AMA1 has no detectable effect on parasite development in the mosquitoes. The capacity of these salivary gland sporozoites to invade cultured hepatocytes was then tested. After sporozoite incubation with HepG2 cells in vitro, a similar proportion of AMA1ko GFP+ versus AMA1+ GFP- parasites is found in the input sporozoites and in hepatic schizonts developing inside HepG2 cells 60 h post infection indicating that AMA1 has no role in P. berghei sporozoite infection of hepatocytes.

Additional information
The infectivity of AMA1KO sporozoites was also tested in vivo. Intravenous injection into mice of as few as 500 AMA1KO/AMA1+ sporozoites or HepG2 cell released hepatic merozoites is sufficient to generate blood-stage parasite populations containing AMA1KO parasites, demonstrating that parasites can complete a life cycle without producing AMA1. Moreover, injection into mice of only 50 AMA1KO/AMA1+ infected erythrocytes is also sufficient to produce AMA1KO-containing blood-stage populations. However, attempts of cloning AMA1KO parasites were unsuccessful. This is likely due to the slower increase in parasitemia of AMA1KO parasites, delaying the emergence of an AMA1KO population that is eventually cleared by the mouse immune system before being detectable. Nonetheless,  the formal hypothesis cannot be ruled out that AMA1KO parasites cannot be cloned because they require soluble AMA1 secreted from the AMA1+ counterparts. The authors state: 'however, this hypothesis of AMA1 as an essential diffusible factor appears unlikely, as AMA1KO growth is observed after co-injection of less than 50 blood stages and 500 sporozoites in the whole animal'.

Other mutants
See RMgm-10 for unsuccessful attempts to disrupt P. berghei ama1
See RMgm-684 for a 'Flp/FRT conditional knock-out mutant' of AMA1. In this mutant the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence AMA1 expression specifically in sporozoites. The phenotype analyses of this mutant indicated that AMA1 is not essential for sporozoite infectivity to liver cells. 'AMA1-silenced' sporozoites showed normal infectivity (traversal and invasion) to hepatocytes in vitro and in vivo. 'AMA1-silenced' sporozoites were not able to produce blood stage infections, indicating the essential role of AMA1 for invasion of (liver) merozoites.