Characterization of SnROP9, a rhoptry protein homologue of Sarcocystis neurona that is expressed in lifecycle stages lacking rhoptry organelles

Proteins released by the club-shaped, apically located, specialized secretory organelles called rhoptries play an essential role in host cell invasion and intracellular survival of apicomplexans. Sarcocystis neurona, the apicomplexan responsible for equine protozoal myeloencephalitis (EPM), lacks rhoptries in its asexual developmental stages, viz., merozoites and schizonts. Nevertheless, rhoptry protein (ROP) homologues were detected in the S. neurona transcriptome and proteome, and SnROP9 was particularly abundant. In this study, we performed in vitro assays to characterize SnROP9 and determine its expression in the merozoite and schizont stages. SnROP9 is a 351 amino acids long protein with two consensus rhoptry protein cleavage motifs. Partition and secretory assays confirmed that SnROP9 is a soluble protein secreted into the excretory-secretory fraction. The total lysate of S. neurona merozoites revealed the full-length protein at ∼38 kDa and two additional peptides at ∼30 kDa and 25 kDa, consistent with its cleavage by a rhoptry processing enzyme. In the schizont stages, the presumed processed SnROP9 peptides migrated differently than in the merozoite and appeared as doublets. In the merozoite, SnROP9 localized predominantly to the apical pole but did not co-localize with the microneme protein, SnMIC10, suggesting that SnROP9 is not trafficked via micronemes, another type of apical secretory organelle. Interestingly, SnROP9 redistributed shortly after the invasion and remained dispersed with a granular appearance throughout the schizont during intracellular development. Despite several attempts, disruption of Snrop9 was unsuccessful, suggesting that there might be an essential role for SnROP9 in S. neurona. Further investigation of SnROP9 and other rhoptry protein homologues will help in better understanding their role in S. neurona biology, particularly in lifecycle stages that lack rhoptry organelles.