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) : 22342550 |
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 |
P. berghei ANKA cl15cy1
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Other information parent line | A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255). |
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The mutant parasite was generated by |
Name PI/Researcher | T. Annoura; C.J. Janse; S.M. Khan |
Name Group/Department | Leiden Malaria Research Group |
Name Institute | Leiden University Medical Center |
City | Leiden |
Country | The Netherlands |
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Name of the mutant parasite |
RMgm number | RMgm-598 |
Principal name | 1345cl1 |
Alternative name | ∆fabb/f |
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 | Normal numbers of salivary gland sporozoites are formed. Sporozoites have a reduced infectivity to mice as shown by a significant delay of blood infections (prolonged prepatent period) after intravenous inoculation of sporozoites. |
Liver stage | Normal numbers of salivary gland sporozoites are formed. Sporozoites have a reduced infectivity to mice as shown by a significant delay of blood infections (prolonged prepatent period) after intravenous inoculation of sporozoites.
Sporozoites showed normal cell traversal, hepatocyte invasion rates and initial stages of intrahepatic development. The formation of merozoites within the liver schizonts was affected (see 'Additional information'). Intravenous injection of 50.000 sporozoites resulted in breakthrough blood infections in the majority (80–100%) of BALB/c mice. All C57BL/6 mice developed a blood stage infection when infected with 50.000 sporozoites. The blood infections show a prolonged prepatency period of 1–2 days as compared to WT parasites. Assuming a P. berghei blood stage multiplication rate of 10× per 24 h this delay to patency indicates a 90–99% reduction in the production and/or infectivity of the Δfabb/f exo-erythrocytic merozoites. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of FABB/F.
Protein (function)
FABB/F is an enzyme of the bacterial like type II fatty acid biosynthesis (FAS-II) pathway. In Plasmodium FAS-II enzymes have been localized to the apicoplast, a nonphotosynthetic plastid organelle of cyanobacterial origin.
FAS-II requires acetyl-Coenzyme A (CoA), which can be converted from pyruvate by the pyruvate dehydrogenase complex. Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA, which is tethered to an acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase (FabD). This produces malonyl-ACP, which, in conjunction with acetyl-CoA, is acted upon by β-ketoacyl-ACP synthase III (Fab H) to form β-ketoacyl-ACP. This precursor enters the FAS-II elongation cycle, mediated by FabB/F (β-ketoacyl-acyl-carrier-protein (ACP) synthase), FabG (β-ketoacyl-ACP reductase), FabZ/A (β-hydroxyacyl-ACP dehydratase), and FabI (trans-2-enoyl-ACP reductase). These four FAS-II enzymes iteratively catalyze the addition of two carbon chains to a growing fatty acyl carbon chain via condensation, reduction, dehydration, and reduction steps, respectively.
Phenotype
The phenotype analyses show that FABB/F is not essential for blood stage development, mosquito stage development and initial infection of the liver. The results indicate an important (but not essential role) in the formation of infective liver stage merozoites demonstrating the importance of FASII pathway for synthesis of fatty acids during late liver stage development. Intravenous injection of 50.000 sporozoites resulted in breakthrough blood infections in the majority (80–100%) of BALB/c mice. All C57BL/6 mice developed a blood stage infection when infected with 50.000 sporozoites. The blood infections show a prolonged prepatency period of 1–2 days as compared to WT parasites. Assuming a P. berghei blood stage multiplication rate of 10× per 24 h this delay to patency indicates a 90–99% reduction in the production and/or infectivity of the Δfabb/f exo-erythrocytic merozoites.
Additional information
Liver stages of Δfabb/f developed into mature forms as shown by qRT-PCR analysis and immuno-fluorescence microscopy. During in vitro liver stage development, the Δfabb/f parasites were morphologically similar to WT parasites as judged by immuno-fluorescence microscopy. However, schizonts showed a significantly lower level of expression of the merozoite surface protein 1 (MSP1); at 48 h post infection (hpi) only 18% of the Δfabb/f schizonts strongly expressed MSP1 whereas 39% of WT parasites were MSP1-positive (p < 0.001) and this increased to 54% in WT and 37% in Δfabb/f at 54 hpi (p = 0.01). The normal morphology of maturing Δfabb/f liver stages and expression of MSP1 parasites is different from the phenotype reported for P. yoelii parasites lacking expression of FabB/F (RMgm-183), where schizonts show clear signs of degeneration and absence of MSP1 expression.
Other mutants
RMgm-599: An independent P. berghei mutant lacking expression of FabB/F.
RMgm-183: A P. yoelii mutant lacking expression of FabB/F.
RMgm-180: A P. yoelii mutant expressing myc tagged FABI
RMgm-181: A P. yoelii mutant expressing myc tagged FABZ
RMgm-182: A P. yoelii mutant expressing myc tagged FABG
RMgm-184: A P. yoelii mutant lacking expression of FABz
RMgm-197: A P. berghei mutant lacking expression of FABI |