Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene disruption,
Introduction of a transgene,
Introduction of a transgene
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 28525314 Reference 2 (PMID number) : 33347893 |
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 820cl1m1cl1 (RMgm-164)
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Other information parent line | P. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517). |
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The mutant parasite was generated by |
Name PI/Researcher | Santos JM, Janse CJ, Mair GR |
Name Group/Department | Parasitology, Department of Infectious Diseases |
Name Institute | University of Heidelberg Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite |
RMgm number | RMgm-4165 |
Principal name | Δlimp-a; Δlimp-b |
Alternative name | 2091cl2m5; 2090cl2m6 |
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 | Normal ookinete production/numbers. Oocyst numbers per mosquito were reduced below 50%. The number of oocyst-derived midgut sporozoites per oocyst remained unchanged; ookinete development and oocyst sporulation therefore do not require LIMP. Δlimp sporozoites did not significantly accumulate in the haemolymph, but showed a 10-fold reduction in salivary gland invasion. |
Sporozoite | Normal ookinete production/numbers. Oocyst numbers per mosquito were reduced below 50%. The number of oocyst-derived midgut sporozoites per oocyst remained unchanged; ookinete development and oocyst sporulation therefore do not require LIMP. Δlimp sporozoites did not significantly accumulate in the haemolymph, but showed a 10-fold reduction in salivary gland invasion. |
Liver stage | Δlimp sporozoites did not significantly accumulate in the haemolymph, but showed a 10-fold reduction in salivary gland invasion. Δlimp salivary gland sporozoites did not establish exoerythrocytic forms (EEFs) inside Huh7 hepatoma cells and were not infectious to mice. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of LIMP and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.
Protein (function)
The gene comprises six exons and five introns (this organisation is conserved throughout the genus) and encodes a protein of 110 amino acids (aa) with a 22 aa long signal peptide and a molecular weight of 13 kDa. Ab initio protein structure predictions indicate that LIMP (I23 to G110) consists of three beta sheets opposed to two a-helices. LIMP is highly conserved among the various Plasmodium species; similarly short proteins are present in related api-complexan parasites, where the homology is focused on a 22 amino acid proline-rich region adjacent to the signal peptide.
Phenotype
Normal ookinete production/numbers. Oocyst numbers per mosquito) were reduced below 50%. The number of oocyst-derived midgut sporozoites per oocyst remained unchanged; ookinete development and oocyst sporulation therefore do not require LIMP. Δlimp sporozoites did not significantly accumulate in the haemolymph, but showed a 10-fold reduction in salivary gland invasion. Δlimp salivary gland sporozoites did not establish exoerythrocytic forms (EEFs) inside Huh7 hepatoma cells and were not infectious to mice.
These results indicate a role for LIMP during mosquito midgut colonisation by the ookinete, and a crucial function for the protein during salivary gland invasion, but not for development; neither ookinete nor sporozoite formation were affected by the absence of LIMP.
In addition evidence is presented for the following:
- Δlimp sporozoites are impaired in hepatocyte transmigration, adhesion and invasion and do not glide
- Transmission electron microscopy (TEM) studies of Δlimp sporozoites revealed no ultrastructural defects in key components of the gliding motility machinery. The arrangement of the plasma membrane (PM), inner membrane complex (IMC), sub-
pellicular microtubules as well as secretory vesicles (micronemes and rhoptries) was unchanged
Additional information
The analyses a GFP-tagged version of LIMP (RMgm-4166) showed the following:
'To assess experimentally when limp is translated, we tagged the endogenous gene at the C terminus with GFP, thus leaving the fusion under the transcriptional control of the native promoter. In this mutant, limp::gfp is the only source for LIMP. We found no protein expression in asexual stage parasites or gametocytes; limp::gfp is translated in the ookinete stage and its expression is visible in crystalloids (transient and putative storage organelles of the ookinete) and the surface; the protein produced a faint ‘dusting’ on the surface of midgut and salivary gland sporozoites. In agreement with previously published results limp mRNA was not detected in asexual blood stages; mRNA is however present in gametocytes (while protein is not) and in ookinetes (where protein is present); this is consistent with limp being translationally repressed and maternally provided to the developing ookinete. Western blot analysis of ookinete material confirmed expression of a GFP fusion protein of the expected size. Parasites expressing LIMP::GFP showed no defects in liver stage development in vitro nor during transmission to the rodent host by mosquito bite'.
GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite.
Additional information from Egarter et al 2020 (PMID: 33347893):
We observed no translation of limp in sexual precursor cells (gametocytes), despite the transcript being readily detected by RT-PCR in this life cycle stage. Using parasite clones expressing GFP-tagged translational repressors DOZI (a homolog of yeast DHH1 DEAD- box RNA helicase) and CITH (a homolog of yeast repressor of translation initiation protein SCD6), we identified limp mRNA to be associated with both proteins by RNA- immunoprecipitation (RIP) followed by RT-PCR detection of the bound transcript.
The decrease of oocyst production (50%) could result from defects in ookinete development, motility, adhesion or invasion, but the underlying cause is not clear. In order to test whether their motility was affected, we performed in vitro ookinete cultures from gametocyte-infected blood. Knock-out parasites produced apparently normal ookinetes as evaluated by differential interference contrast (DIC) microscopy ; they showed no defect in gliding motility. We next tested the ability of in vitro-cultured ookinetes to infect the mosquito vector in a controlled standard membrane feeding assay (SMFA). The gene deletion mutant reproduced the 50 % reduction in oocyst numbers. To characterize the knock-out’s failure to produce high oocyst numbers in more detail, we finally quantified ookinetes in the blood meal after 24 h of four independent SMFA. Therefore, midguts from infected mosquitoes were collected in RPMI and the blood meal isolated; the number of retained ookinetes was quantified via haemocytometer. We found the reduction in oocyst numbers to correlate with an accumulation of ookinetes in the mosquito blood meal bolus. On average, we observed an 82% increase in retained knock-out ookinetes when compared to the wildtype controls, indicating a role for LIMP in attachment/traversal/invasion of the mosquito midgut by the ookinete.
In conclusion limp-depleted zygotes develop into motile ookinetes with apparent normal morphology, and yet display a defect in escaping the mosquito blood meal and reaching the midgut basal lamina where they can transform into the sporozoite-producing oocyst stage
Other mutants
See PF3D7_1207300
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