Abstract
Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.
Highlights
Tsetse flies (Glossina spp.) are obligate vectors of pathogenic African trypanosomes throughout 37 countries in sub-Saharan Africa [1]
Our novel paratransgenic expression system can be applied to study the function of other microRNAs and how they regulate disease transmission in tsetse and other insect systems
Sequencing of the transformation plasmid from several bacterial clones confirmed their identity as either Sgm3xant-microRNA 275 (miR275) or SgmScr-275. These findings indicate that recSodalis was successfully internalized by tsetse cardia and midgut cells where they were protected from the antibacterial effects of gentamicin
Summary
Tsetse flies (Glossina spp.) are obligate vectors of pathogenic African trypanosomes throughout 37 countries in sub-Saharan Africa [1] These parasites belong to the genus of Trypanosoma, which includes the important human pathogens T. b. Free VSG is transiently internalized by cells of tsetse’s PM-producing cardia ( known as proventriculus) [9,10] This process reduces the expression of genes that encode PM associated proteins and digestive enzymes, and modulates the expression of several microRNAs, including a drastic reduction in the expression of tsetse microRNA 275 (miR275) [10]
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