Rickettsial CspA proteins and host microRNAs in exosome formation and pathogenesis

Abstract Rickettsioses are devastating human infections that present major public health concerns and challenges. Microvascular endothelial cells (ECs) are the primary targets of Rickettsia infection; vascular dysfunction in the brain and/or lungs can lead to fatal outcomes. Despite the global distribution and climate change-related emergence of Rickettsia spp., there are sizable knowledge gaps in our understanding of the pathogenic mechanisms of rickettsioses. Using diverse sources of EC-derived exosomes (Exos) collected during R. parkeri infection, we have previously shown that: i) the endothelia can efficiently uptake Exos in vivo; ii) the functions of Exos seem to be linked to their RNA cargos; iii) microvasculopathy-related microRNA (miR23a, miR30b) are selectively sorted into Exos (using infected human umbilical vein EC); iv) only miR23a-27a-24 cluster and miR30b, but not other tested ones, are selectively enriched in Exos (using infected human dermal microvascular EC); and v) exosomal targeting of miR23a during infection ameliorates microvascular dysfunction. While common sequence motifs seem to be shared exclusively among the miR23a cluster and miR30b, the selective enrichment mechanisms remain unclear. We have recently detected rickettsial cold shock protein A (CspA) in Exos, in association with miR27a. Our findings led to the central hypothesis that rickettsial CspA functions as exosome-specific RNA-binding protein (exoRBP) for miR23a/27a/24/30b, regulating selective enrichment of these detrimental exosomal miRs in R. parkeri infection. Building upon our published and established in vitro and in vivo systems, we will test our hypothesis via two independent specific aims. Aim 1 will determine if R. parkeri CspA can act as an exoRBP to selectively enrich exosomal miR23a/27a/24/30b during infection. We will first use cell-free Exo-biogenesis assays with recombinant CspA protein to assess its functional role during miR23a/27a/24/30b loading in Exos. We will then use a miR27a-knockout cell model to examine exosomal expression levels of CspA in complex with miR27a following infection. Aim 2 will assess the biological outcomes in recipient ECs following exposure to pathogenic Exos, or modified Exos with either ectopic expression or deletion of rickettsial CspA. Such complementary gain-vs-loss function approaches will ensure meaningful outcomes for our functional studies. Our research goal is to provide novel and supportive evidence for an emerging paradigm of rickettsial virulence, namely rickettsial proteins can function as exoRBPs for selective package of specific miRs into infected host cell-derived Exos. Research for exosome-based intercellular communications among infected and bystander ECs in the context of vascular dysfunction is highly significant not only for rickettsioses, but also for other vascular-targeted bacterial and viral infections.