RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5230
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1459300; Gene model (P.falciparum): PF3D7_1246200; Gene product: actin I (ACT1; actin1)
Details mutation: Different mutants are described with different point mutations in (subdomains of) actin 1
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 19 September 2022, 14:50
  *RMgm-5230
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35998188
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4478
Other information parent lineThe mutant contains a modified/mutated actin-1 gene locus. It contains the wild-type actin 1 gene under control of the actin 1 5'-UTR whereas the 3'-UTR is from the dhfs gene. In addition, the yfcu::hdfr selection cassette is located in the 3'-UTR region.

This mutant is used as an 'actin-1' recipient line for the introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild-type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC
The mutant parasite was generated by
Name PI/ResearcherYee M, Frischknecht F, Douglas RG
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-5230
Principal namesee below
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteATET/SEPQ developed into mature ookinetes in a manner suggesting no defects with this mutant in early mosquito infection. While development remained unaffected, ATET/SEPQ had highly reduced numbers of moving ookinetes. Other mutants showed normal oocyst production, except for A272S(see below).
OocystWith the exception of A272S, which showed slightly reduced oocyst levels, individual mutants displayed similar oocyst loads to the wild-type control experiment and thus proceeded normally through the initial stages of mosquito infection.
The ATET/SEPQ mutant showed highly reduced total oocyst numbers.
SporozoiteSingle actin mutant sporozoites were impaired in salivary gland invasion, yet only the subdomain 2 single mutant exhibited aberrant motility.
Actin mutants are less transmissible due to deficiency in salivary gland invasion.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
Different mutants are generated and described that contain a modified/mutated actin-1 gene locus. 

While some regions of Plasmodium actin 1 are relatively well conserved (such as subdomain 1), regions in subdomains 2 and 3 contain some of the most divergent stretches of sequence when compared to canonical actins. We selected particular amino acid residues for more detailed study based on three criteria: 1) the Plasmodium actin residue had a striking change in chemical properties compared to their corresponding vertebrate counterpart; 2) the corresponding vertebrate counterpart is conserved across vertebrates and isotypes suggesting critical conservation; and 3) the divergent residues are present in key contact sites either within filaments or potentially towards actin binding proteins. Using these criteria, we earmarked seven divergent residues for mutation: N41H, P42Q (both found on the D-loop of subdomain 2), K270M, A272S, T277E (found on or near the H-plug of subdomain 3), E308P and T315Q (found on the exterior on subdomain 3). While some of these residues do not have direct bonding to neighboring monomer residues, they could mediate intramolecular interactions and/or mutation of these residues to vertebrate counterparts could provide alternative contacts, either by modifying loop dynamics or the vertebrate equivalent forming contacts that do not occur in the native Plasmodium filament, and thus alter filament dynamics. We employed our two-step selection approach to transfect parasites in the blood stages of the life cycle of P. berghei and assess if or where phenotypic consequences occur. We previously attempted the mutations P42Q and K270M in P. berghei but could not replace the wild-type gene, indicating that these mutants confer important interactions that are essential for parasite blood stage development. 

The mutants are generated using the an 'actin-1' recipient line (RMgm-4478) for the introduction of mutated forms of actin-1. Constructs to integrate mutated forms of actin 1 integrate by double homologous integration at the 5'- and 3'-UTR regions of actin-1 thereby replacing the wild-type copy of actin 1 and the yfcu::hdfr selection cassette. These mutants are selected by negative selection using 5-FC

Protein (function)
Actin, a cytoskeletal protein, has many diverse functions in eukaryotic cells ranging from roles in cell motility, cell division, vesicle trafficking to functions in cell signaling and regulation of transcription. A critical property of actin is its ability to form filamentous polymers (F-actin), and a plethora of proteins are involved in the highly dynamic regulation of F-actin formation . Actins are highly conserved proteins that often exist in multiple isoforms in the eukaryotic cell and their expression is regulated both spatially and temporally during development. Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself.

The actin monomer has a highly conserved structure that consists of four subdomains and a central nucleotide (adenosine triphosphate [ATP], adenosine diphosphate with inorganic phosphate [ADP + Pi], or adenosine diphosphate alone [ADP]) binding cleft. Actin possesses the ability to self-assemble from monomers (G-actin) to form filaments (F-actin), which in turn can form higher order filamentous structures. Particular regions in the actin subdomains, such as the hydrophobic plug (H-plug) of subdomain 3 and the highly flexible DNAse I-binding loop (D-loop) of subdomain 2, as well as the nucleotide state, have been implicated as major contributors to the formation and stability of filaments

Plasmodium actin only forms short filaments of approximately 100 nm in length, has a noncanonical filament structure that is dynamically unstable, displays slow polymerisation yet rapid depolymerisation rates, and is regulated by a highly reduced set of predicted actin binding proteins (ABPs)(compared to actins of other/higher eukaryotes). Such altered properties are crucial for intracellular parasite growth and efficient parasite motility.

Phenotype
For the remaining individual mutations in subdomains 2 and 3, as well as a quadruple subdomain 3 mutant (a combined mutant of A272S, T277E, E308P and T315Q; termed ATET/SEPQ), we were able to obtain transfectants possessing the desired residue change(s). Remarkably, these demonstrated no obvious defects in blood stage asexual growth demonstrating that any alteration of actin dynamics mediated by these changes does not manifest at this stage. 

ATET/SEPQ developed into mature ookinetes in a manner suggesting no defects with this mutant in early mosquito infection. While development remained unaffected, ATET/SEPQ had highly reduced numbers of moving ookinetes. Other mutants showed normal oocyst production, except for A272S(see below)

With the exception of A272S, which showed slightly reduced oocyst levels, individual mutants displayed similar oocyst loads to the wild-type control experiment and thus proceeded normally through the initial stages of mosquito infection. The ATET/SEPQ mutant showed highly reduced total oocyst numbers.

Single actin mutant sporozoites were impaired in salivary gland invasion, yet only the subdomain 2 single mutant exhibited aberrant motility.
 
Additional information
Evidence is presented that: Actin mutations have different consequences on hemolymph sporozoite actin localization 

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1459300
Gene Model P. falciparum ortholog PF3D7_1246200
Gene productactin I
Gene product: Alternative nameACT1; actin1
Details of the genetic modification
Short description of the mutationDifferent mutants are described with different point mutations in (subdomains of) actin 1
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationActin 1 ORF replacement constructs were generated as described before. Briefly, a pair of complementary primers of approximately 25 to 30 base pairs containing the mutation(s) of interest were designed. These mutagenesis primers (primers 1–10) were utilized to amplify the sequencing plasmid containing the codon-modified actin 1 ORF. After confirming that these amplicons were of the correct sizes via agarose gel electrophoresis, 1.5 μL of DpnI restriction endonuclease was directly added (New England Biolabs, #R0176S (20 U/μL)) into the remaining 15 μL PCR product. This reaction mix was incubated at 37°C/ 2 h to digest the parental methylated template. Next, 5 μL of this DpnI-digested PCR product was transformed into competent E. coli and the mutation verified via both restriction enzyme digestion and sequencing. The mutated ORFs were subsequently cloned into transfection vector Pb238 via BamHI and XbaI restriction sites. The transfection construct was linearised with SalI and PmlI, transfected into the actin recipient line (RMgm-4478) and integration selected by negative selection using 5-fluorocytosine (1 mg/ml in drinking water) [36,79]. This rendered a line free of the selection cassette and contained the desired change in actin sequence. Isogenic parasites from the transfection mixture of integrants and resistant recipient line was obtained by limiting dilution.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6