Babesia divergensegress from host cells is orchestrated by essential and druggable kinases and proteases

SummaryA unique aspect of apicomplexan biology is the requirement for egress from and invasion into host red blood cells (RBCs). The cellular mechanisms and molecular mediators of RBC egress and invasion remain poorly characterized inBabesiaspp., a group of parasites of veterinary importance and emerging cause of zoonotic disease. Through the use of video microscopy, transcriptomics, and chemical genetics we have implicated signaling, proteases and gliding motility in egress and/or invasion byBabesia divergens. We developed CRISPR/Cas9 and two inducible knockdown systems to perform a genetic screen of putative mediators of egress. We found that proteases ASP2 and ASP3 are required for invasion, and the latter is also required for egress. Strikingly, parasites continue to replicate intracellularly in the absence of the protein kinases, PKG or CDPK4, indicating that they are required for exit from the replication cycle and egress. These essential molecules present druggable targets forBabesia spp. All together we have established a molecular framework for the spread of infection through host RBCs, with egress ofB. divergensmore closely resemblingT. gondiithan the more evolutionarily relatedPlasmodiumspp.HighlightsEgress inBabesia divergensrequires host cell lysis and parasite motilityTranscriptomics can be used to identify egress and invasion proteinsKnockdown of the proteases, ASP2 and ASP3, inhibit egress and invasionInhibition of PKG or CDPK4 signaling results in continued intracellular replication