Paratransgenic manipulation of tsetse miR275 alters the physiological homeostasis of the fly’s midgut environment

Tsetse flies are vectors of parasitic African trypanosomes (Trypanosoma spp.). Current disease control methods include fly-repelling pesticides, trapping flies, and chemotherapeutic treatment of infected people. 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 tsetse’s midgut. It protects the epithelial cells from the gut lumen content such as food and invading trypanosomes, which have to overcome this physical barrier to establish an infection. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse’s gut lumen early during the infection establishment. The VSG molecules are internalized by the fly’s PM-producing cardia, which results in a reduction in tsetse miR275 expression and a sequential molecular cascade that compromises the PM integrity. In the present study, we investigated the role(s) of miR275 in tsetse’s midgut physiology and trypanosome infection processes by developing a paratransgenic expression system. We used tsetse’s facultative bacterial endosymbiont Sodalis glossinidius to express tandem antagomir-275 repeats (or miR275 sponge) that constitutively reduce miR275 transcript abundance. This paratransgenic system successfully knocked down miR275 levels in the fly’s midgut, which consequently obstructed blood digestion and modulated infection outcomes with an entomopathogenic bacteria and with trypanosomes. RNA sequencing of cardia and midgut tissues from the 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 housing symbiotic bacteria. Author Summary Tsetse flies transmit African trypanosomes, which are the parasites that cause sleeping sickness in human in sub-Saharan Africa. When tsetse ingests a blood meal containing trypanosomes, the expression level of a microRNA (miR275) decreases in the fly’s gut. This process results in a series of events that interrupt the physiological homeostasis of the gut environment. To further understand the function of miR275 in tsetse fly, we genetically modified a tsetse’s native bacterial symbiont, reintroduced the genetically modified bacterium back into the fly, and successfully knocked down the miR275 expression in tsetse’s midgut. These ‘paratransgenic’ flies (which house genetically modified bacteria) presented impaired digestive processes and were highly susceptible to infection with trypanosomes. Lastly, we discovered that miR275 regulates tsetse secretory pathways. 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.

9 137 oligos were subjected to restriction endonuclease treatment by SpeI and Sbfi at 37 o C for 2 h. 138 The oligos were then ligated into pgRNA using T4 DNA ligase (NEB), and the constructs were 139 propagated in E. coli DH5a cells. All purified plasmid constructs were sequenced at Yale's Keck 140 Sequencing Laboratory to confirm their structure. The purified DNA plasmids were electroporated into wild-type S. glossinidius morsitans 145 (Sgm WT ) as described previously (33). Two recSodalis strains were used in this study: 1) Sgm 3xant-146 miR275 , which encodes 3xant-miR275, and 2) the miR275 scrambled control (Sgm Scr-275 ) ( Table 1).
147 In brief, 25 mL of log-phase Sodalis cells (OD 600 = 0.3~0.5; SmartSpec Plus spectrophotometer; 148 Bio-Rad, Hercules, CA) were washed consecutively in 25 mL, 1 mL and 1 mL 10% sterile pre-149 chilled glycerol. After the three washes, the Sodalis cell pellets were resuspended in 50 L . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint 150 sterile 10% glycerol. Each 50 L of cell mixture was mixed with 1 or 2 L (~100 ng) of plasmid 151 DNA and subjected to electroporation (voltage, 1.9 kV; capacitance, 25 uF; resistance, 200 152 omega). After electroporation, the recSodalis cells were immediately placed in 5 mL BHI 153 medium for overnight recovery at 26 o C, 10% CO 2 . The recovered cells were then plated on BHI 154 plates supplemented with 10% bovine blood, and transformants were selected with ampicillin 155 (50 g/mL). After a 1-week incubation, transformants were selected for PCR and sequencing.
156 After the sequence was confirmed, a single recSodalis colony was grown in BHI medium for 157 future experiments.

159 2.3 Establishment of paratransgenic tsetse flies
160 To generate paratransgenic tsetse flies, two groups of teneral female flies (newly emerged 161 unfed adults) were given two consecutive blood meals (separated by 1 day) containing either 162 Sgm 3xant-miR275 or Sgm Scr-275 (10 6 CFU/mL each in the first two blood meals) and ampicillin (50 163 g/mL). After a third blood meal (no recSodalis, no ampicillin), 8-day old paratransgenic flies 164 were used in the experiments described below. All plasmid constructs, as well as recSodalis 165 strains and paratransgenic tsetse lines, are summarized in Table 1.

167 2.4 Gentamicin exclusion assay and quantification of recSodalis
168 Gentamicin is unable to cross the eukaryotic cell wall and hence only kills extracellular bacteria 169 (34). Cardia and midgut tissues were dissected from 8-day old paratransgenic and incubated in 170 sterile 0.85% NaCl supplemented with 100 g/mL gentamicin. Controls were incubated in the 171 sterile NaCl in the absence of gentamicin. Tissues were agitated on a shaking platform at room . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 265 Details about sample sizes and statistical tests used for data analyses in this study are indicated 266 in the corresponding figure legends.
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279
We performed gentamicin exclusion assays to confirm that the recSodalis successfully 280 invaded tsetse cardia and midgut cells. Gentamicin cannot penetrate eukaryotic cell 281 membranes, and thus treatment with this antibiotic effectively eliminates the extracellular 282 bacteria but leaves the intracellular population intact (34). We incubated separate cardia and 283 midgut tissues dissected from 8-day old paratransgenic flies in either gentamicin (treatment) or 284 PBS (control). Tissues were subsequently rinsed, homogenized, and plated on BBHI plates 285 supplemented with ampicillin. We recovered 214 ( 54.0) and 9.7x10 5 ( 9.6x10 4 ) gentamicin-286 resistant CFU from the cardia and midgut tissues, respectively (Fig. 1B). Sequencing of the 287 transformation plasmid from several bacterial clones confirmed their identity as either Sgm 3xant-288 miR275 or Sgm Scr-275 . These findings indicate that recSodalis was successfully internalized by tsetse . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint 289 cardia and midgut cells where they were protected from the antibacterial effects of gentamicin.
290 Additionally, significantly more recSodalis cells were present within midgut cells than cells of 291 the cardia organ. We similarly quantified the Sgm 3xant-miR275 and Sgm Scr-275 present in the no 292 gentamicin control groups (cardia, 684  90, p = 0.002; midgut, 2.0x10 6  1.1x10 5 , p < 0.0001) 293 (Fig. 1B), and found that 31% and 49% of recSodalis present in the gut were intracellular within 294 cardia and midgut tissues, respectively. These data also indicated that our recSodalis 295 successfully reside within tsetse's gut at a density similar to that of indigenous Sgm WT in age-296 matched flies (23). Thus, we demonstrated that recSodalis successfully colonized tsetse's gut 297 where they reside within cells that comprise the fly's cardia and midgut tissues.

298
To test the binding efficacy of the antagomirs expressed by 3xant-miR275 to tsetse's 299 mature miR275, we performed a dual luciferase reporter assay. We cloned the 3xant-miR275 300 construct into the multiple cloning site located in the 3'-UTR region of the reporter gene 301 (renilla) in the psiCheck-2 vector (psiCheck-2 3xant-miR275 ). When miR275 binds to the sponge 302 construct cloned in the 3'UTR region of the reporter gene (which initiates the RNA interference 303 (RNAi) process), we expect the renilla transcript to be degraded, and the renilla Luciferase 304 signal to be decreased. The psiCheck-2 vector also contains a firefly reporter in the expression 305 cassette that is designed to be an intra-plasmid transfection normalization reporter. Thus, the 306 Renilla luciferase signal is normalized to the firefly signal to standardize between different 307 biological samples. We measured luciferase activity in three different experiments: 1) psiCheck-308 2 3xant-miR275 + synthetic miR275 mimic, 2) psiCheck-2 3xant-miR275 + synthetic AllStars Negative 309 Control, and 3) psiCheck-2 3xant-miR275 alone, and we found that the relative luciferase activity 310 (renilla/firefly) was significantly suppressed in experiment 1 compared to experiments 2 and 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Fig. 1C). In other words, in the presence of synthetic 312 miR275 mimic, the luciferase activity was significantly repressed, which indicated that our 313 sponge construct was successful when tested in vitro using an insect cell line. This outcome 314 demonstrated that the miR275 effectively binds to the miR275 sponge and initiates the RNAi 315 process with its associated mRNA.

316
To confirm the knockdown effect of miR275 levels in vivo, we used qPCR to quantify the . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Fig. 2A), thus indicating that blood digestion and/or excretory 335 processes (diuresis) were greatly disrupted in Gmm 3xant-miR275 . 336 We next employed a highly sensitive Serratia infection assay to test whether PM  Table S2. We 372 generated multi-dimensional scaling (MDS) plots to understand the overall gene expression 373 differences between the biological replicates and treatment groups. We found that all three 374 replicates within each treatment group clustered closely together as did all control group 375 replicates (Fig. 3A-B). When comparing gene expression differences in the cardia, we found that 395 Given that our phenotypic analysis indicated that miR275 is involved in blood digestion and PM 396 barrier function (Fig. 2), we first evaluated the DE genes whose products are likely associated 397 with these functions. Among the genes whose putative products have been identified as PM 398 structural proteins through proteomics analysis of the PM (44), we found that tsetse EP, midgut . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint 399 trypsin (GMOY007063) and choline acyltransferase were significantly down-regulated, while 400 serine type endopeptidase (GMOY009757), pro1 and GmmPer12 were up-regulated in 401 Gmm 3xant-miR275 relative to Gmm Scr-275 controls ( Fig. 5A; Table S4). Among the secreted products 402 localized to the PM, we found several digestive enzymes, serine proteases (Sp), trypsin and 403 peptidases for which transcript abundance was significantly reduced in the treatment group 404 ( Fig. 5A; Table S4). The reduction in the production of these gene products may account for the 405 impaired blood digestion we noted in Gmm 3xant-miR275 individuals. The down-regulation of 406 several genes whose products are associated with the PM, such as tsetse EP, midgut trypsin, Sp 407 (GMOY006839), Sp15, and choline acyltransferase, were also noted from trypanosome-infected 408 flies where PM functions were also compromised (10). Tsetse EP protein is localized to the 409 midgut, PM, and hemolymph (45, 46). The gene that encodes this protein is immune 410 responsive, as its expression level was upregulated in response to bacterial challenge (45).

424
With respect to blood digestion processes, we detected 10 transcripts involved in heme 425 binding and detoxification processes that were downregulated in Gmm 3xant-miR275 compared to 426 controls ( Fig. 5B; Table S4). Among these putative products were cytochrome (CYP) P450 427 enzymes, which belong to a superfamily involved in insect metabolism, detoxification and 428 insecticide resistance in many different species (50) (Table S5). In addition, we also . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   Table S5). The ubiquitin ligase and a heme binding protein (GMOY001150) were also down 502 regulated in the cardia of Gmm 3xant-miR275 . Ubiquitin ligase and CYP p450, which are associated 503 with insecticide resistance and metabolism of natural or xenobiotic products in many insect 504 species (70), have been linked to toxin metabolism following a blood meal in An. gambiae (71).
505 CYP p450-4g1 is also DE (FC>2) in response to Tbg infections in the Gmm midgut (49). NOS is 506 responsible for producing cellular nitric oxide, which is trypanocidal (72). NOS expression is 507 down regulated in trypanosome-infected SGs (52) and cardia (10, 73), and VSG-treated cardia 508 as well (11) . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

509
Among the SG preferential genes that are dramatically reduced in Gmm 3xant-miR275 cardia, 510 we detected five that were expressed in the midgut: salivary C-type lectin (GMOY000466), Ag5, 511 secreted peptides (GMOY007065 and GMOY007077) and TTI. However, only the salivary C-type 512 lectin was DE in the midgut and upregulated in Gmm 3xant-miR275 relative to controls. 518 is expressed in the SG (Fig. 7A). We anticipated that the miR275 knockdown effects would be 519 restricted to the gut and not impact gene expression levels in other organs. To confirm this, we 520 investigated whether paratransgenic knockdown of miR275 in tsetse's cardia induces a systemic 521 response that results in the knockdown of these genes in the fly's SGs. We first dissected the SG 522 organ from Gmm 3xant-miR275 paratransgenic flies and tested the miR275 expression levels. We 523 subsequently monitored the expression of Adgf3 (GMOY012374), Adgf5 (GMOY012375) and 524 SGP1 (GMOY012268), which are abundantly expressed in tsetse's SGs (52, 60, 61) and 525 downregulated in Gmm 3xant-miR275 cardia. We found that none of the three SG-preferential genes 526 were significantly reduced in the SG of Gmm 3xant-miR275 individuals despite being significantly 527 down-regulated in the cardia (Fig. 7B-D). These results indicate that the effect of the 528 paratransgenic knockdown is restricted to tsetse's gut tissues where recSodalis reside, and does 529 not impact gene expression at the systemic level.

530
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  568 not been possible. To overcome this impediment, we developed the paratransgenic expression 569 system described herein to constitutively express miR275 sponges in tsetse's gut.

570
We consistently observed three phenotypes that are associated with modified tsetse 571 midgut physiological homeostasis in our Gmm 3xant-miR275 flies compared to Gmm Scr-275 controls.
572 These phenotypes all correlate with the presentation of a structurally compromised PM, and 573 they are similar to the phenotypes that we observed previously when synthetic antagomir-275 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 574 was administrated to tsetse. Specifically, we observed that Gmm 3xant-miR275 flies presented 575 significantly heavier gut weights, significantly higher survival rates upon challenge with an 576 entomopathogen, and significantly stronger vector competence, as compared to Gmm Scr-275 577 controls. Increased midgut weight is indicative of impaired blood meal digestion and/or 578 excretion, and this phenotype was similarly observed following treatment of Ae. aegypti (17) 579 and tsetse (11) with synthetic miR275 antagomir. In hematophagous insects, the PM mediates 580 blood digestion by regulating the flux of digestive enzymes from their site of production in the 581 midgut epithelium into the blood bolus-containing gut lumen (81, 82). Our study also 582 demonstrated that significantly more Gmm 3xant-miR275 flies survive in the presence of 583 entomopathogenic Serratia than do Gmm Scr-275 control flies, further indicating that PM 584 functional integrity is compromised in the former group of flies. Serratia marcescens strain 585 Db11 is an entomopathogenic bacterium (83) that can kill tsetse when provided in the 586 bloodmeal. Specifically, flies with an intact PM fail to immunologically detect Serratia, which 587 allows the bacterium to rapidly proliferate in the gut lumen, translocate into the hemolymph 588 and eventually to kill the tsetse and other insects (7, 10, 11, 83-86). Conversely, when PM 589 structural integrity is compromised, the bacterium is quickly detected by tsetse's midgut 590 epithelium and eliminated by the fly's robust antimicrobial immune response. The Serratia 591 infection assay thus serves as a highly sensitive indicator of tsetse's PM structural integrity (7). 592 Lastly, we observed a higher trypanosome infection prevalence in Gmm 3xant-miR275 flies 593 compared to Gmm Scr-275 controls. This outcome is similar to what observed in flies exposed to 594 anti-PM RNAi (dsRNA targeting pro1, pro2 and chitin synthase) (7) as well as in flies that were 595 provisioned a blood meal containing a purified trypanosome coat protein (sVSG), which . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 597 Taken together, our results confirm that interference with miR275 expression in the cardia and 598 midgut of Gmm 3xant-miR275 flies results in the modified gut environment we noted in this study.

599
Herein we repeatedly observed phenotypes that correspond with a depletion of miR275 600 expression in tsetse's cardia. However, despite these findings, we were unable to quantify a . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.15.435453 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made