Last updated 3 years ago

Disruptability [+]

Species Disruptability Reference Submitter
P. yoelii yoelii 17X
Possible
RMgm-4797 Imported from RMgmDB
P. yoelii yoelii 17X
Refractory
RMgm-5055 Imported from RMgmDB
P. berghei ANKA
Possible
PlasmoGEM (Barseq) PlasmoGEM
P. falciparum 3D7
Refractory
USF piggyBac screen (Insert. mut.) USF PiggyBac Screen

Mutant phenotypes [+]

Species Stage Phenotype Reference Submitter
P. yoelii yoelii 17X Asexual
No difference
RMgm-4797 Imported from RMgmDB
P. yoelii yoelii 17X Gametocyte
Difference from wild-type
RMgm-4797
(somewhat) reduced production of gametocytes; normal exflagellation
Imported from RMgmDB
P. berghei ANKA Asexual
No difference
PlasmoGEM (Barseq) PlasmoGEM
P. falciparum 3D7 Asexual
Egress defect
35862778
\'CDC50B is not required for GCα expression or trafficking but is crucial for optimal cGMP synthesis required for egress. GCα has a key role in egress as the source of cGMP required for PKG activation (18). Having determined that CDC50B interacts with GCα, we next investigated whether CDC50B also has a role in parasite egress. To do this, we first compared the egress kinetics of mature DMSO- and RAP-treated CDC50B-HA:loxP schizonts by monitoring the appearance in culture supernatants over time of proteolytically processed forms of the PV protein serine repeat antigen 5 (SERA5), which acts as a proxy for egress due to its release upon schizont rupture (35). As shown in Fig. 4A, these experiments revealed a marked reduction in the rate of egress over the sampling period in RAP-treated CDC50B-HA:loxP parasites compared to that of control DMSO-treated counterparts. Densitometric quantitation of data from three independent experiments indicated that CDC50B null schizonts undergo ~50% less egress than WT controls when sampled over a 2-h period (see Fig. S1 in the supplemental material). This was not due to a delay in schizont development, since microscopic examination of Giemsa-stained DMSO- and RAP-treated CDC50B-HA:loxP schizonts showed no detectable delay in parasite maturation, and analysis of DNA content by flow cytometry indicated no significant differences between formation of DMSO- and RAP-treated CDC50B-HA:loxP schizonts (Fig. 4A, lower panel). We further investigated the egress phenotype of CDC50B null parasites using a flow cytometry time course to assess the production of new rings by highly synchronized DMSO- and RAP-treated CDC50B-HA:loxP schizonts. We observed that while the number of new rings formed in the CDC50B null population between 45 and 53 h postinvasion was reduced, by 69 h postinvasion there was no statistically significant difference in ring parasitemia between the control and CDC50B null parasites (Fig. 4B). Microscopic examination of these newly generated ring stage parasites showed that those derived from the DMSO-treated parasites appeared more mature than those derived from the RAP-treated cultures (Fig. 4B, inset boxes), suggesting a delay to invasion in the latter. Together, the data indicate that CDC50B null parasites exhibit an extended erythrocytic life cycle, resulting in a delay to egress after schizont maturation but with no overall reduction in new ring stage formation in the following cycle.\'
Theo Sanderson, Francis Crick Institute

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