Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene tagging
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 28115054 |
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 |
Not applicable
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Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | Klug D, Frischknecht F |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | Heidelberg University Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite |
RMgm number | RMgm-4119 |
Principal name | gfp-trp1 |
Alternative name | |
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 sporozoite formation in oocysts. Normal egress of sporozoites from oocysts. (Slightly) reduced salivary gland sporozoites. Normal infectivity to mice (by mosquito bite). No GFP expression. |
Sporozoite | Normal sporozoite formation in oocysts. Normal egress of sporozoites from oocysts. (Slightly) reduced salivary gland sporozoites. Normal infectivity to mice (by mosquito bite). No GFP expression. |
Liver stage | Normal sporozoite formation in oocysts. Normal egress of sporozoites from oocysts. (Slightly) reduced salivary gland sporozoites. Normal infectivity to mice (by mosquito bite). No GFP expression. |
Additional remarks phenotype | Mutant/mutation
A mutant expressing an N-terminal GFP-tagged version of TRP1 (contained an N-terminal GFP placed in-between the signal sequence and the remaining ORF)
Protein (function)
Thrombospondin-related protein 1 (TRP1) has a single α-thrombospondin repeat (αTSR) domain. The αTSR is especially interesting because this domain is believed to be involved in protein-protein interactions, as shown for the TSRs of thrombospondin-1. Proteins containing TSRs are widespread among animals and protozoans and are mostly extracellular or secreted proteins. At least one TSR is contained within all proteins of the TRAP-family, including MTRAP, which was recently found to be important for gametocyte egress from red blood cells. The general domain composition of TRP1 shows many similarities to that of the thrombospondin related anonymous protein (TRAP). It can be found in all Plasmodium species and has a highly conserved core region that includes the TSR and the TMD. Both TRP1 and TRAP contain a signal peptide (SP), a TSR, a transmembrane domain (TMD) and a cytoplasmic tail domain (CTD). TRP1 lacks the penultimate tryptophan (W) that has been shown to be important for TRAP function as well as the von Willebrandt factor like A-domain that is present at the N-terminus of TRAP and important for invasion. Due to the overall similarity to TRAP-family proteins, TRP1 was grouped together with TRAMP, TRSP and SSP3 in the family of TRAP-related proteins that have a potential cytoplasmic tail domain (CTD) but lack the conserved tryptophan.
Phenotype
In the paper the the phenotypes of the following mutants have been analysed:
- trp1(-) - a mutant lacking expression of TRP (RMgm-4117)
- trp1(-)mCh (RMgm-4118) - a mutant lacking expression of TRP and expressing mCherry under control of the trp promoter
- gfp-trp1 (RMgm-4119) - a mutant expressing an N-terminal GFP-tagged version of TRP1 (contained an N-terminal GFP placed in-between the signal sequence and the remaining ORF) (no GFP expression)
- gfp-trp1ΔN (RMgm-4120) - a mutant expressing a mutated form of TRP1 where the sequence after the signal peptide until the start of the TSR (549 aa) was removed. In addition it contained an N-terminal GFP placed in-between the signal sequence and the remaining ORF (weak GFP expression)
- gfp-trp1ΔC (RMgm-4121) - a mutant expressing a mutated form of TRP1 where the last 41 amino acids of the open reading frame, corresponding to the CTD was removed. In addition it contained an N-terminal GFP placed in-between the signal sequence and the remaining ORF (no GFP expression).
- trp1-gfp (RMgm-4121) - a mutant expressing a C-terminal GFP-tagged version of TRP1 (strong GFP expression)
Combined these phenotype analyses showed the following:
- Parasites lacking TRP1 failed to migrate within oocysts and did not egress, suggesting that TRP1 is a vital component of the events that precede intra-oocyst motility and subsequently sporozoite egress and salivary gland invasion.
- TRP1 expression starts in young oocysts based on expression of mCherry under control of the TRP1 promoter (see also below)
- Sporozoites collected from oocysts are motile
- Sporozoites collected from oocysts and injected intravenously in mice show a normal infectivity to mice
- More hemolymph sporozoites were detected in the gfp-trp1ΔN and gfp-trp1ΔC mutants than in mutants lacking expression of TRP1. However, in both mutants no salivary gland sporozoites were found
- Gfp-trp1ΔN sporozoites are more capable of egressing from oocysts than gfp-trp1ΔC sporozoites.
- Evidence is presented that the N-terminus of TRP1 is important for oocyst egress and that both the N- and the C-terminus are essential for salivary gland invasion. Intriguingly, the N-terminus of TRP1 is the least conserved part of the protein across Plasmodium species.
- Normal sporozoite formation and egress but lower salivary gland invasion of sporozoites expressing a C-terminal GFP-tagged version of TRP1
- The C-terminal GFP-fusion protein of the full-length TRP1 localized strongly at the periphery of the oocyst. In sporozoites TRP1-GFP appears to localize in what might be a subset of micronemes or a different organelle that does not extend all the way to the front.
- Evidence is presented that that TRP1 is transported through the ER, is post-translationally processed and can accumulate at the periphery of the parasite on its rear end.
Additional information
Other mutants
See above
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