| Liver stage | The PbGFPEXP1ΔCT parasites display a significant reduction in the number of infected hepatocytes in vitro analyzed 24 and 48 h post-infection (hpi), as well as in the size of the EEFs replicating intra-hepatically at these time points, relative to their corresponding controls. Additionally, our data show that the number of PbEXP1ΔCT merosomes formed at 72 hpi is markedly decreased compared to that observed for PbWT control parasites.
Quantification of hepatic infection in mice, at 42 h after intravenous infection with sporozoites, showed that the parasite burden in the livers of PbGFPEXP1ΔCT-infected mice was drastically reduced compared with that of mice infected with the corresponding control parasite lines. Importantly, when infection was allowed to proceed to the ensuing erythrocytic stage following sporozoite injection, parasites were detected in the blood of all PbWT spz-infected mice, whereas all the mice infected with PbEXP1ΔCT spz remained blood-stage negative. |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal(CT)-domain truncated version of PbEXP1.
Protein (function)
Exp1 (HEP17) is a protein that localizes to the parasitophorous vacuole membrane (PVM) of blood stages and liver stages
Pb/PfEXP1’s topology includes an N-terminal (NT) domain (aa 1-75/1-79) that harbors a classical signal peptide (aa 1-23); a putative catalytic domain that includes the Pf Arg70 residue and is presumed to be responsible for GST activity in blood stage parasites; an internal hydrophobic region typical of transmembrane anchor sequences (aa 75-97/79-101); and a C-terminal (CT) domain (aa 97-166/101-162) that includes a C1 (aa 103-136/107-134) and a C2 (aa 137-166/135-162) region, as defined in (Sa et al., 2017). In both blood and liver parasite stages, EXP1 is proposed to be integrated into the PVM with the N-terminus facing the lumen of the PV, and the C-terminus exposed to the host cell cytosol
Phenotype
Reduced oocyst production; Reduced (salivary gland) sporozoite formation (evidence is presented that sporozoites are not affected in exit from oocysts and invasion of salivary glands).
The PbGFPEXP1ΔCT parasites display a significant reduction in the number of infected hepatocytes in vitro analyzed 24 and 48 h post-infection (hpi), as well as in the size of the EEFs replicating intra-hepatically at these time points, relative to their corresponding controls. Additionally, our data show that the number of PbEXP1ΔCT merosomes formed at 72 hpi is markedly decreased compared to that observed for PbWT control parasites.
Quantification of hepatic infection in mice, at 42 h after intravenous infection with sporozoites, showed that the parasite burden in the livers of PbGFPEXP1ΔCT-infected mice was drastically reduced compared with that of mice infected with the corresponding control parasite lines. Importantly, when infection was allowed to proceed to the ensuing erythrocytic stage following sporozoite injection, parasites were detected in the blood of all PbWT spz-infected mice, whereas all the mice infected with PbEXP1ΔCT spz remained blood-stage negative.
Additional information
A similar mutant was made in P. berghei ANKA wild type background.
In order to dissect the functional role of different domains of PbEXP1, we attempted to generate modified versions of this protein bearing a truncated version of its N- and C-terminal domains. Despite multiple attempts, we were unable to generate a modified version of PbEXP1 lacking the NT domain (PbEXP1ΔNT; RMgm-4656), suggesting that it is essential for blood stage development of Pb parasites. However, transgenic Pb parasite lines expressing a CT domain-truncated version of PbEXP1 were successfully generated in both the PbANKA (PbWT) and PbGFPcon (PbGFP) parental parasite lines, yielding their PbEXP1ΔCT and PbGFPEXP1ΔCT mutated counterparts, respectively, and showing that Pb blood stages can develop in the absence of the PbEXP1 CT domain. Successful deletion of the CT domain in the PbEXP1ΔCT parasites was confirmed by Western blot using two different anti-EXP1 antibodies that bind either to epitopes present on the full length PbEXP1 protein or to epitopes present only on the protein’s C-terminus.
Evidence is presented that in PbGFPEXP1ΔCT parasites EXP1 is not at the PVM but is remains located in the PV
Other mutants |
*RMgm-4655
