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. |