Nicastro, C. | Search bioRxiv

bioRxiv2022

Functional characterization of the thrombospondin-related paralogous proteins rhoptry discharge factor 1 and 2 unveils phenotypic plasticity in Toxoplasma gondii rhoptry exocytosis

To gain access to the intracellular cytoplasmic niche essential for their growth and replication, apicomplexan parasites such as Toxoplasma gondii rely on the timely secretion of two types of apical organelles named micronemes and rhoptries. Rhoptry proteins are key to host cell invasion and remodelling, however, the molecular mechanisms underlying the thight control of rhoptry discharge are poorly understood. Here, we report the identification and functional characterization of two novel T. gondii thrombospondin-related proteins implicated in rhoptry exocytosis. The two proteins, already annotated as MIC15 and MIC14, were renamed rhoptry discharge factor 1 (RDF1) and rhoptry discharge factor 2 (RDF2) and found to be exclusive of the Coccidia class of apicomplexan parasites. Furthermore, they were shown to have a paralogous relationship and share a C-terminal transmembrane domain followed by a short cytoplasmic tail. Immunofluorescence analysis of T. gondii tachyzoites revealed that RDF1 presents a diffuse cytoplasmic localization not reminiscent of any know subcellular compartment, whereas RDF2 was not detected. Using a conditional knockout approach, we demonstrated that RDF1 loss caused a marked growth defect. The lack of the protein did not affect parasite gliding motility, host cell attachment, replication and egress, whereas invasion was dramatically reduced. Notably, while RDF1 depletion did not result in altered microneme exocytosis, rhoptry discharge was found to be heavily impaired. Interestingly, rhoptry secretion was partially reversed by spontaneous upregulation of the RDF2 gene in knockdown parasites grown under constant RDF1 repression. Collectively, our results identify RDF1 and RDF2 as additional key players in the pathway controlling rhoptry discharge. Furthermore, this study unveils a new example of compensatory mechanism contributing to phenotypic plasticity in T. gondii.

microbiology↗

bioRxiv2017

A Model for Genome-First Care: Returning Secondary Genomic Findings to Participants and Their Healthcare Providers in a Large Research Cohort

BackgroundResearch cohorts with linked genomic data exist, or are being developed, at many research centers. Within any such \"sequenced cohort\" of more than 100 participants, it is likely that there are participants with previously undisclosed risk for life-threatening monogenic diseases that could be identified with targeted analysis of their existing data. Identification of such disease-associated findings are not usually primary to the enrollment research goals. At Geisinger Health System, MyCode(R) Community Health Initiative (MyCode) participants represent one such large sequenced cohort. Since 2013, MyCode participants in discovery research have been consented for secondary analysis of their existing research genomic sequences to allow delivery of medically actionable findings to them and their healthcare providers. This return of genomic results program was developed to manage an anticipated 3.5% of MyCode participants who will receive clinically confirmed genomic variants from an approved gene list out of more than 150,000 total participants. Risk-associated DNA sequences alone without any clinical parameter, prompt \"genome-first\" follow-up encounters.\n\nMethodsThis article describes our process for generating clinical grade results from research-based genomic sequencing data, delivering results to patients and their providers, facilitating targeted clinical evaluations of patients and promoting cascade testing of at-risk relatives. We also summarize our early data about the results generated during this process and our ability to contact patients and their providers to disclose the information.\n\nResultsThis process has been used to generate 343 results on 339 patients. 93% of patients with a result have been successfully contacted about their results as evidenced by direct interaction about their result with the research team or a healthcare provider. 222 healthcare providers have been notified of a result on one or more patient through this result delivery process.\n\nConclusionsHere we describe the existing GHS model to deliver genomic data into the electronic medical record and the clinical interactions that are prompted and supported. Elements of this genome-first care model can be applied in other healthcare settings and in national efforts, such as \"All of Us\", that wish to establish programs for returning genomic results to research participants.

genomics↗