Molecular Characterization of Differentially Expressed Genes During Glossina Morsitans Morsitans and Trypanosoma\Congolense Interactions

Abstract

African trypanosome, Trypanosoma congolense parasite transmitted by tsetse flies (Glossina spp) cause Animal African Trypanosomiasis in (AAT or Nagana) in animals. The parasite undergo cyclical ( development in the tsetse vector and transform to become metacyclics which .are forms infective to animals. The molecular events underpinning this development process and tsetse responses to infection in cardia and proboscis is underexplored due to challenges of obtaining enough biological materials, access to tsetse colony and analysis complexes of such studies. This study characterized differentially expressed genes during Glossina morsitans morsitans and T. congolense interactions in the cardia and proboscis of tsetse fly to shed light on the molecular cross-talks between the parasite and the vector during infections in these organs. This study was a laboratory-based and involved use and infection oflaboratory adapted tsetse flies and parasites. Ribonucleic acid was extracted from T.congolense infected male G.. m. morsitans cardia or proboscis (PB) and sequenced in high throughput Illumina Next Generation Sequencing (NGS) platform. EdgeR and CLC-genomics was used to determine differentially expressed transcripts in tsetse and trypanosomes, and functional analysis done using blast2GO, profcom, KEGG, WebGestalt and TrypanoCyc. Reverse genetics (RNAi silencing) was used to functionally characterize the roles of selected peritrophic matrix (PM) peritrophin (Per) 12 and/or 108in the cardia. Immunofluorescent and confocal microscope was used to localize expression of selected parasite protein. Statistical analysis was performed either using R-statistic or GraphPad prism version 7. Cardia-enriched transcripts (n=422) encoded putative proteins functionally associated with peritrophic matrix structure. Upon infection of the cardia, 88transcripts were suppressed while62 were induced. The suppressed transcripts were functionally associated with transport and metabolic process while induced ones with immunity, suggesting enhanced defense mechanism in the cardia against trypanosomes. The RNAi significantly suppressed (51-81%) expression of per transcripts and resulted in significantly higher number of tsetse flies being infected by trypanosome but impeded proliferation of Serratia bacteria (in GmmPer12). Proboscis enriched transcripts (n=668) were predominately associated with muscle tissue, chemo-sensation, chitin-cuticle development as well as mechanoreceptors that monitor blood flow during tsetse feeding and interaction with trypanosomes. Cellular structures associated with muscles and cells in the proboscis were evident by microscopy. Like cardia, more transcripts were suppressed (n= 88) than induced (n=38) by the infection in the proboscis. The induced genes were associated with cell division while those suppressed were associated with metabolic processes, extracellular matrix, ATP-binding and immunity suggesting increased cell and tissue renewal process of host. Oxidative phosphorylation and amino acid metabolism appears to be a major source of energy for the parasite. in both organs. More transcripts were induced (n= 1261) in parasite infecting the cardia than those infecting the proboscis (n=870). Product of cardia induced parasite transcripts were associated with cell signaling, quorum sensing and several transport activities suggesting that the parasites in the cardia scavenge for nutrients and also exhibit social behavior. The parasites in the cardia were putatively covered by Fam50 'GARP', 'iii' and 'i' proteins. The product of proboscis-induced parasite transcripts were associated with nucleosomes, cytoplasm and membrane-bound organelles suggesting increased cell division. Overall, these findings suggest that cardia is immunologically active organ with cells that produce PM proteins that also play critical role in modulating trypanosome infection outcome. The parasites also express genes encoding putative cell surface proteins and proteins associated with parasite differentiations that may regulate T. congolense developmental processes in tsetse fly. The proboscis is a muscular organ with chemosensory and mechanosensory capabilities. This study provides insight into vector-parasite interactions and points to potential molecular targets that can be exploited in downstream search for disease transmission control initiative.