| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene mutation
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 29253313
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| MR4 number |
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| Parent parasite used to introduce the genetic modification |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17XNL |
| 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 | Steel RWJ, Kappe SHI |
| Name Group/Department | Center for Infectious Disease Research |
| Name Institute | formerly Seattle Biomedical Research Institute |
| City | Seattle |
| Country | USA |
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| Name of the mutant parasite |
| RMgm number | RMgm-4407 |
| Principal name | Py s4/celtos(ctrp) |
| 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 | Normal gametocyte production. A partial but incomplete rescue of the number of oocysts per midgut was seen in Py S4/CelTOS(CTRP) parasites. |
| Oocyst | Normal gametocyte production. A partial but incomplete rescue of the number of oocysts per midgut was seen in Py S4/CelTOS(CTRP) parasites. While Py S4/CelTOS(CTRP) parasites had fewer oocysts per midgut than Py WT, the percent of Py S4/CelTOS(CTRP) infected midguts was not significantly lower than Py WT. |
| Sporozoite | Normal gametocyte production. A partial but incomplete rescue of the number of oocysts per midgut was seen in Py S4/CelTOS(CTRP) parasites. While Py S4/CelTOS(CTRP) parasites had fewer oocysts per midgut than Py WT, the percent of Py S4/CelTOS(CTRP) infected midguts was not significantly lower than Py WT. The rescue of midgut infection by the Py S4/CelTOS(CTRP) parasite (compared to CelTOS null mutants) translated into robust salivary gland sporozoite loads, although the number of sporozoites was lower than in Py WT. |
| Liver stage | Injection of 100 sporozoites of each parasite line revealed that Py S4/CelTOS(CTRP) sporozoites were significantly less infectious than Py WT sporozoites (4 out of 11 mice developed blood stage infection with a 1 day delayed prepatent period). When infection was delivered by five mosquito bites, all Py WT infected mice but none of the Py S4/CelTOS(CTRP) infected mice developed blood stage infection. Increasing the challenge to 15 infectious bites resulted in 47% of Py S4/CelTOS(CTRP) infected mice.
Cell traversal by Py S4/CelTOS(CTRP) sporozoites was significantly reduced compared to Py WT. Cell invasion by Py S4/CelTOS(CTRP) sporozoites was comparable to Py WT. There was no difference between the number of liver stage schizonts 48 hours after infection with Py WT and Py S4/CelTOS(CTRP) parasites, excluding a role for S4/CelTOS during successful in vitro infection.
Analysis of live-microscopic imaging data revealed that significantly fewer Py S4/CelTOS(CTRP) than Py WT sporozoites exhibited normal gliding motility. Of the Py S4/CelTOS(CTRP) sporozoites that did glide, a greater proportion detached from the gliding surface prematurely, although they moved with slightly greater velocity while attached. |
| Additional remarks phenotype | Mutant/mutation
In the 'promoter swap mutant': the promoter of CelTOS replaced with the ookinete-specific ctrp promoter (PY17X_0415800; circumsporozoite- and TRAP-related protein).
Protein (function)
CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted. It plays a role in migration of ookinetes through mosquito midgut cells and traversal of sporozoites through hepatocytes
Phenotype
CelTOS has essential/important role in traversal of ookinetes through the midgut epithelium. In order to analyse the role of CelTOS in sporozoites in more detail the promoter-swap mutant Py s4/celtosctrp was generated, where celtos is under control of the ookinete-specific ctrp promoter, resulting in rescue of the ookinete phenotype in mutants lacking celtos.
Normal gametocyte production. A partial but incomplete rescue of the number of oocysts per midgut was seen in Py S4/CelTOS(CTRP) parasites. While Py S4/CelTOS(CTRP) parasites had fewer oocysts per midgut than Py WT, the percent of Py S4/CelTOS(CTRP) infected midguts was not significantly lower than Py WT. The rescue of midgut infection by the Py S4/CelTOS(CTRP) parasite (compared to CelTOS null mutants) translated into robust salivary gland sporozoite loads, although the number of sporozoites was lower than in Py WT.
Injection of 100 sporozoites of each parasite line revealed that Py S4/CelTOS(CTRP) sporozoites were significantly less infectious than Py WT sporozoites (4 out of 11 mice developed blood stage infection with a 1 day delayed prepatent period). When infection was delivered by five mosquito bites, all Py WT infected mice but none of the Py S4/CelTOS(CTRP) infected mice developed blood stage infection. Increasing the challenge to 15 infectious bites resulted in 47% of Py S4/CelTOS(CTRP) infected mice.
Cell traversal by Py S4/CelTOS(CTRP) sporozoites was significantly reduced compared to Py WT. Cell invasion by Py S4/CelTOS(CTRP) sporozoites was comparable to Py WT. There was no difference between the number of liver stage schizonts 48 hours after infection with Py WT and Py S4/CelTOSCTRP parasites, excluding a role for S4/CelTOS during successful in vitro infection.
Analysis of live-microscopic imaging data revealed that significantly fewer Py S4/CelTOSCTRP than Py WT sporozoites exhibited normal gliding motility. Of the Py S4/CelTOS(CTRP) sporozoites that did glide, a greater proportion detached from the gliding surface prematurely, although they moved with slightly greater velocity while attached.
Additional information
We were unable to detect S4/CelTOS protein in Py S4/CelTOS(CTRP) salivary gland sporozoites by either indirect immunofluorescence assay (IFA) or western blot, while expression of other adhesion and motility motor proteins such as CSP, TRAP and MTIP were unaffected. Collectively these data demonstrate that the CTRP promoter swap strategy substantially rescued the deficit in midgut infection associated with the complete S4/CelTOS knockout, yielding robust salivary gland sporozoite numbers that were deficient in S4/CelTOS.
We sought to confirm the role for S4/CelTOS during gliding for other Plasmodium parasite species, and to this end we generated a Pb S4/CelTOS(CTRP) line (RMgm-4408). As for Py S4/CelTOS(CTRP), we observed that gliding trails for Pb S4/CelTOS(CTRP) were shorter than Pb WT.
Other mutants |
*RMgm-4407
