FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

Abstract

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…Parasitemia was determined by flow cytometry using a FACSCalibur cytometer: the signal emitted by green fluorescent protein-parasites was collected in the FL-1 channel and data were analysed using FlowJo software. The targeting plasmid was constructed by cloning three PCR products into the plasmid described by Lacroix et al 62 , which carries the human dihydrofolate reductase expression cassette that confers the resistance to WR99210 drug. The pre 5 0 -untranslated region of Pbsub1 (736 bp) DNA fragment was amplified using primers 5 0 -AAGCTTTAAATAAATCACCAAATATTAAA TTGG-3 0 and 5 0 -GCGGCCGCATGTGTGTTATATAAGTGATTATTCTT TAAGC-3 0 , and cloned into the plasmid using HindIII and NotI restriction sites (underlined).…”

A Key Role for Plasmodium Subtilisin-like SUB1 Protease in Egress of Malaria Parasites from Host Hepatocytes

“…The P. berghei NK65 UIS4/FLP and GFP-expressing clone, which expresses the FLP under the control of the uis4 promoter (27), was transfected to obtain the SUB1/Cond conditional clones. Anopheles stephensi (Sda500 strain) mosquitoes were fed on infected mice 3-5 days after emergence and kept at 21°C and 70% humidity as described (11).…”

“…Southern Blot, PCR, and RT-PCR Analysis-Southern blot analyses were carried out as described (27,32). Genomic DNA from P. berghei parental and SUB1/Cond clones 5 and 22 was extracted using the QIAamp DNA blood kit (Qiagen); digested with NcoI, XbaI, or AccI; separated by gel electrophoresis; and transferred overnight to Hybond N ϩ membrane (GE Healthcare).…”

“…berghei Transfection and Cloning-The targeting pSUB1/ FRT plasmid was constructed by cloning three PCR products into a plasmid that carries two FRT sites and the human dihydrofolate reductase expression cassette (27,28): (i) a 736-bp fragment upstream from the Pbsub1 locus; (ii) a 3479-bp DNA fragment encompassing the 5Ј FRT site, 1177 bp corresponding to the 5Ј-untranslated region (UTR) of Pbsub1, the complete PbSUB1 open reading frame (1799 bp), and 503 bp corresponding to the Pbsub1 3Ј-UTR; and (iii) a 673-bp fragment downstream from the Pbsub1 locus. The latter was amplified using the oligonucleotides 5Ј-CCCGGGCATTTGGTGTATATTAT-TGTTTTTCTTG-3Ј and 5Ј-GGTACCTCAATGTGCATTT-ATTGTTATGC-3Ј and was cloned first in the plasmid using SmaI and KpnI restriction sites (underlined sequences).…”

“…Moreover, hepatic merozoites express MSPs and are able to invade erythrocytes. In this work, we show that SUB1 is expressed by hepatic merozoites, and, by generating sub1-null liver stages using FLP/flippase recognition target (FRT)-mediated conditional mutagenesis in P. berghei (27,28), we show that SUB1-deficient hepatic merozoites are unable to egress from infected hepatocytes and to establish a blood infection. Together, these data show that SUB1 plays a key role during the last phase of Plasmodium biological development into host hepatocytes, qualifying this enzyme as an attractive target of intervention strategies aiming at preventing as well as curing infection in humans.…”