Abstract
Host cell invasion by apicomplexan parasites is governed by coordinated and sequential exocytosis of specialized apical organelles: micronemes and rhoptries. Our knowledge on exocytosis mechanisms of these organelles remains incomplete. Together with Ciliata and Dinoflagellata, apicomplexan parasites belong to the Alveolata superphylum. Although morphologically different, Alveolata share several features, including the presence of secretory organelles. Previous studies in the Ciliata Paramecium tetraurelia showed that the exocytosis of their defensive extrusive organelles, called trichocysts, could be triggered upon stimulation. Studies on Paramecium mutants defective for trichocysts exocytosis (called Nd for “non-discharge”), led to the identification of essential components of the trichocysts-membrane fusion machinery. Previously, our team showed that some of these Nd genes have orthologs in T.gondii and P.falciparum (Nd6 and Nd9), which are involved in rhoptry secretion and invasion. This phylogenetic conservation was further explored and prompted the identification of T.gondii orthologs in Tetrahymena as new Nd genes. These results support the hypothesis of an Alveolate conserved mechanism for rhoptry secretion.Given that in Ciliata, Nd genes and trichocysts biogenesis-related genes are similarly transcribed, we then identified genes co-regulated with the Tetrahymena Nd6 gene and conserved in Apicomplexa. From this screen, we showed that 2 genes of T.gondii encoding Nd11 and Nd12 are involved in invasion and their the knock-down leads to loss of rhoptry secretion. Nd11 and Nd12 are cytosolic proteins and possess multiple transmembrane domains, protein-protein interaction and sugar binding domains. Mass spectrometry and co-immunoprecipitation assays showed a reciprocal interaction between Nd11 and Nd12, and identified 2 other unknown proteins, TgME49_277910 and TgME49_247115 both also shown to be involved in rhoptrie secretion. In conclusion, by combining a phylogenetic with a transcriptomic approach we identified a new complex, essential for rhopty secretion, in T.gondii and new effectors of exocytosis in apicomplexan parasites.