Additional remarks phenotype | Mutant/mutation
The mutant lacks expression 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 indicate indicates 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 (see also 'Additional information').
Additional information
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.
Evidence is presented that the percentage of developing liver stages surrounded by a UIS4-positive parasitophorous vacuole membrane (PVM) was significantly reduced in mutant parasites compared to wild type liver stages. Mutant parasites had a 33% and 43% decrease compared to wild type parasites of UIS4 positive staining at 2 hours and 6 hours, respectively. Evidence is presented that UIS4 can serve as a substrate to rhomboid proteases. UIS4 belongs to the eTRAMP protein family that contain a conserved transmembrane domain with sequence similarity to known rhomboid substrates.
Electron microscope analyses indicate that while mutant sporozoites are capable of productively invading host cells with the formation of a PVM, a fraction of them have a defect in the subsequent PV expansion and modification in early development.
Analysis of a mutant expressing a (N-terminal) HA-tagged version of PyROM1 (RMgm-660) confirmed expression of the protein in blood stage schizonts and in salivary gland sporozoites. Immunofluorescence analyses showed co-localisation with micronemal (MAEBL), rhoptry (RON4) and endoplasmic reticulum (BiP) markers.
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.
Initially, a single crossover homologous recombination strategy was used to disrupt the endogenous pyrom1 locus. Successful disruption of the pyrom1 gene was confirmed by Southern blot and RT-PCR (not entered into the RMgm database). Because single crossover disruptants can revert to wildtype, another deletion mutant was created using a gene replacement vector to exchange the endogenous pyrom1 gene for a pbDHFR/TS-GFP cassette by double crossover homologous recombination (described here).
Other mutants
RMgm-176: A P. berghei mutant lacking expression of ROM1.
RMgm-187: Unsuccessful attempts to disrupt rhomboid protease ROM4 of P. berghei.
RMgm-660: A mutant expressing a (N-terminal) HA-tagged version of PyROM1 |