Summary

RMgm-660
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0935500; Gene model (P.falciparum): PF3D7_1114100; Gene product: rhomboid protease ROM1 (ROM1)
Name tag: a triple hemaglutinin tag (3xHA)
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 3 December 2013, 20:49
  *RMgm-660
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21909259
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherI. Medina Vera; K. Kim
Name Group/DepartmentDepartments of Medicine and of Microbiology and Immunology
Name InstituteAlbert Einstein College of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-660
Principal namePyROM1-HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageROM1-HA expression in asexual blood stages (see 'Additional remarks phenotype')
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteROM1-HA is readily detected in salivary gland sporozoites with very minimal expression in midgut sporozoites.
Liver stageDuring sporozoite invasion into Hepa 1–6 cells, ROM1-HA is observed in developing EEFs at four hours post invasion (see 'Additional remarks phenotype')
Additional remarks phenotype

Mutant/mutation
The mutant expresses  a (N-terminal) HA-tagged version of rhomboid protease ROM1.

Protein (function)
Rhomboid proteins are intra-membrane proteases that play a role in multiple processes. They belong to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. The Plasmodium genome encodes a total of 8 rhomboid proteases (ROM1, 3, 4, 6, 7, 10; Dowse, TJ and Soldati, D, 2005, Trends Parasitol 21, 254-58) that show stage-specific expression patterns and includes proteins upregulated in gametocytes and sporozoites. ROM1 is expressed in both the blood stages and mosquito stages (sporozoites). ROM1 was localized to organelles of the apical complex of merozoites and was shown to be able to cleave different adhesins of all invasive stages (merozoites, ookinetes, sporozoites).

Phenotype
Phenotype analyses of synchronized blood stages showed ROM1-HA protein expression is lowest in ring stages (3 hours post invasion) and increases during the course of development with a peak in schizont stages. In sporozoite stages, ROM1-HA is readily expressed in salivary gland sporozoites with very minimal expression in midgut sporozoites.

Phenotype analyses of a P. yoelii mutant lacking expression of ROM1 (RMgm-659) indicate  a mild attenuation of of blood stages of mutant parasites. Evidence is presented that lack of ROM1 expression does not affect development of oocysts, sporozoite release into the hemolymph,  invasion of salivary glands and sporozoite motility and invasion of hepatocytes. During development of the mutant liver stages, the number of infected hepatocytes significantly decreased during the first 24 hours compared to wild type liver stages and evidence is presented that mutant liver stages are affected in the formation of a parasitophorous vacuole membrane (PVM)  and that UIS4, a PVM protein, can serve as a substrate to rhomboid proteases.

Additional information
Evidence is presented that ROM1-HA co-localizes with the microneme adhesin, pyMAEBL and with the rhoptry neck protein RON4 at the apical end of merozoites within mature schizonts by immunofluorescence. Although localization overlapped more consistently with pyMAEBL than RON4, pyROM1 shows partial co-localization with both markers. Some localization overlap is observed between PyROM1-HA and the endoplasmic reticulum (ER) marker BiP. PyROM1-HA was found in a speckled intracellular pattern in salivary gland sporozoites (day 14) that partially overlaps with localization of PyMAEBL and PyUIS4. Some co-localization is observed with BiP in salivary gland sporozoites. During sporozoite invasion into Hepa 1–6 cells, ROM1 remains prominent with a speckled pattern diffusely dispersed throughout the sporozoite. Expression of ROM1-HA is observed in developing EEFs at four hours post invasion with intracellular localization throughout the length of the parasite that is distinct from the localization of the sporozoite surface antigen, circumsporozoite protein (CSP)

The sequence of P. yoelii rhomboid 1 (pyrom1) obtained consists of 4 exons and 3 introns, encompassing two annotated genes py00729 and py00728. Based on topology predictions (TMHMM and HMMTOP), PyROM1 has seven transmembrane domains with the canonical rhomboid catalytic serine motif (GASTS) found within transmembrane domain four and a conserved histidine found within transmembrane domain six. It has an N-terminal tail of 52 amino acids that includes the conserved microneme targeting motif YPHY and a very short carboxy terminal tail.

Amplification of Pyrom1 cDNA from synchronized erythrocytic stages shows modest expression, with greatest expression in schizont (S) stages. There is a 10-fold increased expression in midgut (MG) sporozoites and a 20-fold increased expression in salivary gland (SG) sporozoites relative to expression levels of schizont stages.

A mutant lacking expression of ROM1 has also been generated in P. berghei (RMgm-176). Phenotype analyses of this mutant indicated that ROM1 plays distinct roles during P. berghei development. It is non-essential but appears to play a role in blood stages, the transformation of ookinetes into oocysts and in the establishment of infection of the liver by the sporozoite. P. berghei  ROM1 is not required for sporozoite invasion of the salivary glands.

Other mutants
RMgm-659: A P. yoelii mutant lacking expression of ROM1
RMgm-176:  A P. berghei mutant lacking expression of ROM1.
RMgm-187: Unsuccessful attempts to disrupt rhomboid protease ROM4 of P. berghei.


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0935500
Gene Model P. falciparum ortholog PF3D7_1114100
Gene productrhomboid protease ROM1
Gene product: Alternative nameROM1
Details of the genetic modification
Name of the taga triple hemaglutinin tag (3xHA)
Details of taggingN-terminal
Additional remarks: taggingTriple HA tag under control of the pyrom1 5' and 3' regulatory elements.
Commercial source of tag-antibodiesanti-HA mAb clone 3F10 (Roche)
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid Aria
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe spliced open reading frame amplified from P. yoelii 17XNL cDNA was cloned into a vector containing an N-terminal triple HA tag using the EcoRI and NotI restriction sites. The 5' flanking region (FR) was amplified from gDNA (1.5 kb) and was cloned into a pBluescript SK(-) (Stratagene) plasmid using restriction sites ApaI and XbaI to generate pB5'FR. The HApyrom1 fragment was subsequently cloned within the XbaI and Not I sites to generate the pB5'FRHAR1 plasmid. The 3'FR (1 kb) was then cloned using NotI and SacII sites into the pB5'HAR1 plasmid to generate pB5'FRHAR13'FR plasmid. The entire insert containing a 5'FR, an N terminally tagged pyrom1 ORF, and a 3'FR, was then cut out with ApaI and SacII and was cloned into the PL0006 vector that contains the hDHFR selectable marker (Leiden) to ultimately generate the pR1HA knock-in vector. Prior to P. yoelii transfection the pR1HA vector was linearized with the restriction enzyme Aria in order to promote homologous recombination within the 5'FR.

See table S3 in the supporting information for primer sequences used in this study
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