Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut... SHORT COMMUNICATION | Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 114: e190217, 2019 1 5 Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans 1,2 2 3 Larissa F Paranaiba , Alessandra A Guarneri , Ana C Torrecilhas , 1 2 + Maria N Melo , Rodrigo P Soares / Universidade Federal de Minas Gerais, Departamento de Parasitologia, Belo Horizonte, MG, Brasil Fundação Oswaldo Cruz-Fiocruz, Instituto René Rachou, Belo Horizonte, MG, Brasil Universidade Federal de São Paulo, Departamento de Ciências Farmacêuticas, Diadema, SP, Brasil The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host. Key words: Trypanosoma cruzi - triatomines - extracellular vesicles - interaction Chagas disease is caused by the protozoan Trypano- general, EVs are composed of a phospholipid bilayer soma cruzi (Kinetoplastida: Trypanosomatidae). It is es- containing lipids, proteins, glycoconjugates and nucleic (9) timated that more than eight million people are infected acids. The role of EVs during interaction with vectors and 25 million are at risk of acquiring the disease. More is poorly understood. However, it is already known that than 10 thousand people die per year due to complications in another Trypanosomatid, Leishmania major, EVs are of clinical manifestations of the disease. Chagas disease released by promastigotes in the midgut of the sand fly was originally found in the Americas, but recently, due vector and further inoculated together with the parasite (10) to human migration, has expanded to non-endemic coun- in the vertebrate host. Altogether, this inoculum will tries in North America, Europe and Asia. T. cruzi is trans- be important for cell attraction and parasite establish- mitted by triatomines (Reduviidae: Triatominae) includ- ment in the vertebrate host. In this context, T. cruzi EVs ing Triatoma infestans and Rhodnius prolixus, the most have already demonstrated their pro-inflammatory ac- (1,2,3) important vectors in Latin America. tivity during host innate and chronic immune responses. (11,12) To develop and establish infection within the hos- However, no study on the role during the interaction tile environments in the digestive tract of the vec- with triatomine vector was performed. tor and vertebrate hosts, T. cruzi developed a variety Here, we provide evidence that in the digestive tract of of strategies implicating a wide number of molecules. triatomine vetors, T. cruzi EVs were able to functionally (4) Those are important for attachment and internalisa- affect early parasite migration in R. prolixus and had no (5) (6) tion including Tc-85, glycoinositolphospholipids and effect on the number of metacyclics in both vector species. (7) glycosylphosphatidylinositol(GPI)-mucins. Some of Bug2149 cl10 T. cruzi strain (Bug), originally isolated those molecules can be either shed or expressed in the from naturally infected T. infestans (Rio Grande do Sul, (13) surface of extracellular vesicles (EVs). Brazil) was used. Epimastigote forms were cultured EVs are spontaneously released by any cells includ- in liver-infusion tryptose (LIT) supplemented with 15% (8) ing prokaryotic and eukaryotic. Depending on the ori- foetal bovine serum (FBS), 100 µg/mL streptomycin, 100 gin, size and function they can be classified as microves- units/mL penicillin (27ºC and pH 7.2). T. infestans and R. icles, nanoparticles, apoptotic bodies and exosomes. In prolixus used in this study were obtained from a labora- tory colony derived from insects collected in Brazil and Honduras, respectively. Triatomines were reared at 25 ± 1ºC, 60 ± 10% relative humidity and natural illumination (14) as previously reported. Fourth instar nymphs, starved for 30 days after ecdysis, were used in the assays. doi: 10.1590/0074-02760190217 Parasites were grown in LIT medium, washed in Financial support: CNPq, FAPEMIG, FAPESP. hanks’ balanced salt solution (HBSS), centrifuged RPS, AAG and MNM are supported by CNPq and FAPEMIG (PPM-00102-16); LFP is supported by FAPEMIG; ACT is supported by (1000g/10min, 10ºC) and counted. For EVs release, T. FAPESP (2016-01917-3). 5 cruzi in early log phase (1 x 10 parasites/mL) were re- + Corresponding author: rodrigo.pedro@fiocruz.br suspended in LIT medium without FBS and incubated  https://orcid.org /0000-0002-7966-3629 at 28ºC for 2 h. Parasites were fixed and cover slips Received 26 June 2019 Accepted 22 November 2019 were prepared for scanning electron microscopy (SEM) online | memorias.ioc.fiocruz.br | 2 5 Larissa F Paranaiba et al. and transmission electron microscopy (TEM) as pre- alysing the whole slide in optical microscopy. The same (11,12) viously reported. After vesiculation, supernatants nymphs were dissected as described above at 49-50 days (14) were collected, filtered (0.22 μm) and ultra-centrifuged p.i. Sample size was calculated as reported elsewhere. (100,000g/2h, 4ºC). Nanoparticle tracking analysis To test whether or not the data follow a normal dis- (NTA) was performed to determine size, distribution and tribution, the test Kolmogorov-Smirnov was performed. (12) concentration of EVs as reported elsewhere. Acquisi- Data showing a normal distribution were analysed by tions were measured in a Nanosight NS300 instrument t test or analysis of variance (ANOVA). In the case of (Malvern Instruments Ltd, Malvern, UK) equipped with ANOVA, pairwise comparisons were performed by a 405-nm laser and coupled to a charge-coupled device means of Tukey post hoc tests. Non-parametric data (CCD) camera (the laser emitting a 60-mW beam at 405- were analysed by Mann-Whitney test. P < 0.05 was con- nm wavelength). Data were analysed using NTA soft- sidered significant. ware (version 2.3 build 0017). The detection threshold T. cruzi epimastigotes were able to release EVs (Fig. was set to 10. Blur, Min track Length and Min Expected 2A-B). Based on the data of the NTA, EVs exhibited an Particle Size were set to auto. To perform the measure- average size of 223.1 nm (D10 = 143.6; D50 = 245.5 and ments, samples were diluted 1:100 in phosphate-buffered D90 = 264.7) (Fig. 2C) and a mean concentration be- saline (PBS). Readings were taken in triplicates during tween 6.84 x 10 particles/mL (Fig. 2D). Released EVs 30 s at 20 frames per second (three times for each sam- were also subjected to TEM in order to confirm integrity ple), at camera level set to 14 and manual monitoring of and their sizes were within the range detected in NTA temperature (19ºC). (Fig. 3A-D). For functional studies, those vesicles were Citrated rabbit blood was obtained from Centro de mixed to rabbit blood and offered to the triatomines. Criação de Animais de Laboratório (CECAL), Fiocruz, After EVs exposure and infection, T. cruzi migration RJ. Insects were artificially exposed to EVs and parasites in different parts of the digestive tract was compared in two consecutive moments: (i) first day, nymphs were in two periods of infection (21-22 and 49-50 days p.i.). artificially fed on citrated heat-inactivated (56ºC, 30 min) In R. prolixus, there was an increase in the number of rabbit blood containing EVs. Each insect was allowed parasites present in the midgut when compared to the to ingest 20-30 μL of blood (approximately 6.4-9.6 x 10 control group at 21-22 days p.i. (Mann Whitney, p < particles/µL); (ii) second day, the same nymphs were 0.0001; Fig. 4A). In the rectum, however, the number of artificially fed to repletion on citrated heat-inactivated parasites was reduced in the nymphs treated with EVs rabbit blood containing epimastigotes. Those parasites (Mann Whitney, p < 0.0001; Fig. 4A). After 49-50 days were obtained from LIT cultures, washed in PBS and p.i. the number of parasites in the midgut was reduced in resuspended in the rabbit blood at a final concentration both treatments, with no differences between the groups of 1 x 10 parasites/mL. Since each insect could ingest (Mann Whitney, n.s.; Fig. 4B). For T. infestans, no differ- 20-30 mL of blood, the number of epimastigotes would ences in the number of parasites between EVs-exposed range from 2-3 x 10 . Control group was fed on blood nymphs and controls were observed (Mann Whitney, and blood + parasites with similar amounts of the treated n.s.; Fig. 5A-B). At 28 days p.i. the number of metacy- group in the respective days. The midgut and rectum of clis in the urine did not vary among controls and EV ex- a group of nymphs (n = 20) were individually dissected posed insects for both triatomine species (t test, p > 0.05, (Fig. 1) and homogenised in 30 μL of PBS (0.15 M NaCl Fig. 6A-B). All T. infestans nymphs released parasites in at 0.01 M sodium phosphate, pH 7.4) at 21-22 days post urine, but in numbers ~10 fold smaller than those found infection (p.i.). Quantification of parasites was made in in R. prolixus ones. Neubauer chamber. At 28 days p.i., a new feeding with Parasites are known release exosome-like EVs that citrated heat-inactivated rabbit blood was offered for the function as cell-to-cell effectors during the host-parasite (11,12,15,16,17) remaining nymphs (n = 15). Immediately after feeding, interaction. One of the initial studies showed the nymphs were transferred to 1.5 mL plastic tubes and that challenge of BALB/c mice with T. cruzi EVs exacer- (11) the urine produced during diuresis was collected to eval- bated parasite load, heart inf lammation and mortality. uate the percentage of metacyclic forms. 10 µL of each Later, it was demonstrated that T. cruzi could modulated sample were fixed in 10% methanol and stained with Gi- not only the innate but also the acquired immune events emsa®. The numbers of parasites were quantified by an- by activating TLR2, triggering cytokine production, (12,18) MAPKs activation and invasion. It is important to mention that the concentration of vesicles used in those studies ranged from 1-10 µg/mL. Here, pre-feeding with EVs was approximately 5 µg/mL. Although it may be not physiological, this concentration is within a range known to functionally activate vertebrate cells. Howev- er, reports regarding the role of EVs with respect to their invertebrate hosts are unknown. R. prolixus and T. infestans are the most used tri- atomine models due to due to existence of laboratory colonies. Both models were successfully used in our Fig. 1: basic diagrammatic representation of a digestive tract from a procedures and produced the expected infection pattern (14,15,16,17,18,19) triatomine bug. as previously reported. However, the abil- | Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 114, 2019 3 5 Fig. 2: extracellular vesicles (EVs) of Trypanosoma cruzi. (A) Scanning electron microscopy (SEM) of T. cruzi membrane shedding (bars: 1-5 µm), Magnification 32,657 x and (B) Scanning electron microscopy (SEM) of T. cruzi membrane shedding (bars: 1-5 µm) Magnification 80,000 x. Nanoparticle tracking analysis (NTA) (C) and (D) of T. cruzi EVs. Fig. 3: extracellular vesicles (EVs) of Trypanosoma cruzi. Transmis- sion electron microscopy (TEM) of T. cruzi membrane shedding (A) (lower magnification, bar: 200 nm) and (B) (higher magnification, bar: 100 nm). ity of T. infestans to take a blood meal in the artificial system was much lower than that of R. prolixus. Pre- feeding with EVs delayed the early migration of T. cruzi parasites to the rectum (21-22 days p.i.) in R. prolixus. Fig. 4: parasites found throughout the digestive tract of Rhodnius pro- This effect was not observed after 49-50 days p.i., sug- lixus. Dissections at 21-22 (A) and 49-50 (B) days after the infective gesting a transient effect of the EVs during the initial feeding. Each dot represents the quantification of parasites from one days of infection. However, this effect was not detected individual nymph and each horizontal bar corresponds to the median in T. infestans in both periods. The number of metacy- of the group evaluated. clics in EV exposed and controls did not vary for both vectors reinforcing their role only in the initial events of infection. Interestingly, the number of metacyclic try- belongs to the tribe Rhodniini, whereas T. infestans is (20) pomastigotes recovered in the urine of R. prolixus was from the Triatomini tribe. We thus believe that such 10-fold higher than in T. infestans. This result was very differences may be attributed to the species rather than surprising since Bug strain was originally isolated from the amount of ingested blood. Although in our model, T. infestans in Brazil, whereas the population of R. pro- pre-feeding with EVs affected early parasite migration lixus used in this study was from Honduras. R. prolixus only in R. prolixus, their role in transmission was not as- | 4 5 Larissa F Paranaiba et al. Fig. 6: metacyclic trypomastigotes found in the urine of Rhodnius pro- lixus (A) and Triatoma infestans (B) at 28 days p.i. Each dot represents the quantification of parasites in the urine sample of one nymph and Fig. 5: parasites found throughout the digestive tract of Triatoma in- each horizontal bar corresponds to the mean of the group evaluated. festans. Dissections at 21-22 (A) e 49-50 (B) days after the infective feeding. Each dot represents the quantification of parasites from one individual nymph and each horizontal bar corresponds to the median of the group evaluated. 5. Alves MJ, Colli W. Trypanosoma cruzi: adhesion to the host cell and intracellular survival. IUBMB Life. 2007; 59: 274-9. 6. Nogueira NF, González MS, Gomes JE, de Souza W, Garcia ES, sessed. Since the number of total parasites and metacy- Azambuja P, et al. Trypanosoma cruzi: involvement of glycoinosi- clics in later days of infection did not vary, it is not likely tolphospholipids in the attachment to the luminal midgut surface that EVs will affect transmission in triatomines. This is of Rhodnius prolixus. Exp Parasitol. 2007; 116(2): 120-28. different from L. major, where EVs were important for (10) 7. Soares RP, Torrecilhas AC, Assis RR, Rocha MN, Moura e Castro parasite transmission during the sand f ly bite. Despite FA, Freitas GF, et al. Intraspecies variation in Trypanosoma cruzi the presence of EVs, we showed that both vectors were GPI-mucins: biological activities and differential expression of able to develop metacyclics. However, in nature those α-galactosyl residues. Am J Trop Med Hyg. 2012; 87(1): 87-96. parasites are released in the feces and urine and are not 8. Szempruch AJ, Dennison L, Kieft R, Harrington JM, Hajduk SL. inoculated as in Leishmania. Sending a message: extracellular vesicles of pathogenic protozoan In vertebrate cells, it is already known that fusion parasites. Nat Rev Microbiol. 2016. 14(11): 669-75. of EVs is an important mechanism that promotes para- (11,18) 9. Campos JH, Soares RP, Ribeiro K, Andrade AC, Batista WL, site internalisation. Our results suggest that T. cruzi Torrecilhas AC. Extracellular vesicles: role in inflammatory re- EVs could be fusing to the epithelium of the midgut sponses and potential uses in vaccination in cancer and infectious and somehow promoting early parasite retainment in R. diseases. J Immunol Res. 2015; 2015: 832057. prolixus. This effect was transient and did not affect the 10. Atayde VD, Aslan H, Townsend S, Hassani K, Kamhawi S, Ol- number of metacyclics in both vectors. ivier M. Exosome secretion by the parasitic protozoan Leishmania AUTHORS’ CONTRIBUTION within the sand f ly midgut. Cell Rep. 2015; 13(5): 957-67. RPS, AAG, ACT and MNM conceived and planned the 11. Torrecilhas ACT, Tonelli RR, Pavanelli WR, da Silva JS, Schum- experiments; LFP, AAG and ACT performed experiments and acher RI, de Souza W, et al. Trypanosoma cruzi: parasite shed analysed data. All authors wrote and corrected the manuscript. vesicles increase heart parasitism and generate an intense inf lam- matory response. Microbes Infect. 2009; 11(1): 29-39. REFERENCES 12. Nogueira PM, Ribeiro K, Silveira ACO, Campos JH, Martins-Fil- 1. Pérez-Molina JA, Molina I. Chagas disease. Lancet. 2018; ho OA, Bela SR, et al. Vesicles from different Trypanosoma cruzi 391(10115): 82-94. strains trigger differential innate and chronic immune responses. J Extracell Ves. 2015; 4: 28734. 2. Antinori S, Galimberti L, Bianco R, Grande R, Galli M, Corbel- lino M. Chagas disease in Europe: a review for the internist in the 13. Palace-Berl F, Pasqualoto KFM, Jorge SD, Zingales B, Zorzi RR, globalized world. Eur J Intern Med. 2017; 43: 6-15. Silva MN, et al. Designing and exploring active N′-[(5-nitrofuran- 3. Schofield CJ, Dias JC. The Southern Cone initiative against Cha- 2-yl) methylene] substituted hydrazides against three Trypanoso- ma cruzi strains more prevalent in Chagas disease patients. Eur J gas disease. Adv Parasitol. 1999; 42: 1-27. Med Chem. 2015; 96: 330-9. 4. Ferguson MA. The structure, biosynthesis and functions of glyco- sylphosphatidylinositol anchors, and the contributions of trypano- 14. Ferreira RC, Kessler RL, Lorenzo MG, Paim RMM, Ferreira some research. J Cell Sci. 1999; 112(Pt 17): 2799-2809. LDL, Probst CM, et al. Colonization of Rhodnius prolixus gut by | Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 114, 2019 5 5 Trypanosoma cruzi involves an extensive parasite killing. Parasi- 18. Ribeiro KS, Vasconcellos CI, Soares RP, Mendes MT, Ellis CC, tology. 2016; 143(4): 434-43. Aguilera-Flores M, et al. Proteomic analysis reveals different composition of extracellular vesicles released by two Trypano- 15. Torrecilhas AC, Schumacher RI, Alves MJM, Colli W. Vesicles as soma cruzi strains associated with their distinct interaction with carriers of virulence factors in parasitic protozoan diseases. Mi- host cells. J Extracell Ves. 2018; 7(1): 1463779. crobes Infect. 2012; 14(15): 1465-74. 19. Castro DP, Moraes CS, González MS, Ratcliffe NA, Azambuja 16. Marcilla A, Martin-Jaular L, Trelis M, de Menezes-Neto A, Osuna P, Garcia ES. Trypanosoma cruzi immune response modulation A, Bernal D, et al. 2014. Extracellular vesicles in parasitic dis- decreases microbiota in Rhodnius prolixus gut and is crucial for eases. J Extracell Ves. 2014; 1: 1-15. parasite survival and development. PLoS One. 2012; 7(5): e36591. 17. Torró LMP, Moreira LR, Osuna A. Extracellular vesicles in Cha- 20. Lent H, Wygodzinsky PW. Revision of the triatominae (Hemip- gas disease: a new passenger for an old disease. Front Microbiol. tera, Reduviidae), and their significance as vectors of Chagas dis- 2018; 9: 1190. ease. Bull Am Mus Nat Hist. 1979; 163: 125-520. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Memórias do Instituto Oswaldo Cruz Pubmed Central

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans

Memórias do Instituto Oswaldo Cruz, Volume 114 – Dec 13, 2019

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SHORT COMMUNICATION | Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 114: e190217, 2019 1 5 Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans 1,2 2 3 Larissa F Paranaiba , Alessandra A Guarneri , Ana C Torrecilhas , 1 2 + Maria N Melo , Rodrigo P Soares / Universidade Federal de Minas Gerais, Departamento de Parasitologia, Belo Horizonte, MG, Brasil Fundação Oswaldo Cruz-Fiocruz, Instituto René Rachou, Belo Horizonte, MG, Brasil Universidade Federal de São Paulo, Departamento de Ciências Farmacêuticas, Diadema, SP, Brasil The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host. Key words: Trypanosoma cruzi - triatomines - extracellular vesicles - interaction Chagas disease is caused by the protozoan Trypano- general, EVs are composed of a phospholipid bilayer soma cruzi (Kinetoplastida: Trypanosomatidae). It is es- containing lipids, proteins, glycoconjugates and nucleic (9) timated that more than eight million people are infected acids. The role of EVs during interaction with vectors and 25 million are at risk of acquiring the disease. More is poorly understood. However, it is already known that than 10 thousand people die per year due to complications in another Trypanosomatid, Leishmania major, EVs are of clinical manifestations of the disease. Chagas disease released by promastigotes in the midgut of the sand fly was originally found in the Americas, but recently, due vector and further inoculated together with the parasite (10) to human migration, has expanded to non-endemic coun- in the vertebrate host. Altogether, this inoculum will tries in North America, Europe and Asia. T. cruzi is trans- be important for cell attraction and parasite establish- mitted by triatomines (Reduviidae: Triatominae) includ- ment in the vertebrate host. In this context, T. cruzi EVs ing Triatoma infestans and Rhodnius prolixus, the most have already demonstrated their pro-inflammatory ac- (1,2,3) important vectors in Latin America. tivity during host innate and chronic immune responses. (11,12) To develop and establish infection within the hos- However, no study on the role during the interaction tile environments in the digestive tract of the vec- with triatomine vector was performed. tor and vertebrate hosts, T. cruzi developed a variety Here, we provide evidence that in the digestive tract of of strategies implicating a wide number of molecules. triatomine vetors, T. cruzi EVs were able to functionally (4) Those are important for attachment and internalisa- affect early parasite migration in R. prolixus and had no (5) (6) tion including Tc-85, glycoinositolphospholipids and effect on the number of metacyclics in both vector species. (7) glycosylphosphatidylinositol(GPI)-mucins. Some of Bug2149 cl10 T. cruzi strain (Bug), originally isolated those molecules can be either shed or expressed in the from naturally infected T. infestans (Rio Grande do Sul, (13) surface of extracellular vesicles (EVs). Brazil) was used. Epimastigote forms were cultured EVs are spontaneously released by any cells includ- in liver-infusion tryptose (LIT) supplemented with 15% (8) ing prokaryotic and eukaryotic. Depending on the ori- foetal bovine serum (FBS), 100 µg/mL streptomycin, 100 gin, size and function they can be classified as microves- units/mL penicillin (27ºC and pH 7.2). T. infestans and R. icles, nanoparticles, apoptotic bodies and exosomes. In prolixus used in this study were obtained from a labora- tory colony derived from insects collected in Brazil and Honduras, respectively. Triatomines were reared at 25 ± 1ºC, 60 ± 10% relative humidity and natural illumination (14) as previously reported. Fourth instar nymphs, starved for 30 days after ecdysis, were used in the assays. doi: 10.1590/0074-02760190217 Parasites were grown in LIT medium, washed in Financial support: CNPq, FAPEMIG, FAPESP. hanks’ balanced salt solution (HBSS), centrifuged RPS, AAG and MNM are supported by CNPq and FAPEMIG (PPM-00102-16); LFP is supported by FAPEMIG; ACT is supported by (1000g/10min, 10ºC) and counted. For EVs release, T. FAPESP (2016-01917-3). 5 cruzi in early log phase (1 x 10 parasites/mL) were re- + Corresponding author: rodrigo.pedro@fiocruz.br suspended in LIT medium without FBS and incubated  https://orcid.org /0000-0002-7966-3629 at 28ºC for 2 h. Parasites were fixed and cover slips Received 26 June 2019 Accepted 22 November 2019 were prepared for scanning electron microscopy (SEM) online | memorias.ioc.fiocruz.br | 2 5 Larissa F Paranaiba et al. and transmission electron microscopy (TEM) as pre- alysing the whole slide in optical microscopy. The same (11,12) viously reported. After vesiculation, supernatants nymphs were dissected as described above at 49-50 days (14) were collected, filtered (0.22 μm) and ultra-centrifuged p.i. Sample size was calculated as reported elsewhere. (100,000g/2h, 4ºC). Nanoparticle tracking analysis To test whether or not the data follow a normal dis- (NTA) was performed to determine size, distribution and tribution, the test Kolmogorov-Smirnov was performed. (12) concentration of EVs as reported elsewhere. Acquisi- Data showing a normal distribution were analysed by tions were measured in a Nanosight NS300 instrument t test or analysis of variance (ANOVA). In the case of (Malvern Instruments Ltd, Malvern, UK) equipped with ANOVA, pairwise comparisons were performed by a 405-nm laser and coupled to a charge-coupled device means of Tukey post hoc tests. Non-parametric data (CCD) camera (the laser emitting a 60-mW beam at 405- were analysed by Mann-Whitney test. P < 0.05 was con- nm wavelength). Data were analysed using NTA soft- sidered significant. ware (version 2.3 build 0017). The detection threshold T. cruzi epimastigotes were able to release EVs (Fig. was set to 10. Blur, Min track Length and Min Expected 2A-B). Based on the data of the NTA, EVs exhibited an Particle Size were set to auto. To perform the measure- average size of 223.1 nm (D10 = 143.6; D50 = 245.5 and ments, samples were diluted 1:100 in phosphate-buffered D90 = 264.7) (Fig. 2C) and a mean concentration be- saline (PBS). Readings were taken in triplicates during tween 6.84 x 10 particles/mL (Fig. 2D). Released EVs 30 s at 20 frames per second (three times for each sam- were also subjected to TEM in order to confirm integrity ple), at camera level set to 14 and manual monitoring of and their sizes were within the range detected in NTA temperature (19ºC). (Fig. 3A-D). For functional studies, those vesicles were Citrated rabbit blood was obtained from Centro de mixed to rabbit blood and offered to the triatomines. Criação de Animais de Laboratório (CECAL), Fiocruz, After EVs exposure and infection, T. cruzi migration RJ. Insects were artificially exposed to EVs and parasites in different parts of the digestive tract was compared in two consecutive moments: (i) first day, nymphs were in two periods of infection (21-22 and 49-50 days p.i.). artificially fed on citrated heat-inactivated (56ºC, 30 min) In R. prolixus, there was an increase in the number of rabbit blood containing EVs. Each insect was allowed parasites present in the midgut when compared to the to ingest 20-30 μL of blood (approximately 6.4-9.6 x 10 control group at 21-22 days p.i. (Mann Whitney, p < particles/µL); (ii) second day, the same nymphs were 0.0001; Fig. 4A). In the rectum, however, the number of artificially fed to repletion on citrated heat-inactivated parasites was reduced in the nymphs treated with EVs rabbit blood containing epimastigotes. Those parasites (Mann Whitney, p < 0.0001; Fig. 4A). After 49-50 days were obtained from LIT cultures, washed in PBS and p.i. the number of parasites in the midgut was reduced in resuspended in the rabbit blood at a final concentration both treatments, with no differences between the groups of 1 x 10 parasites/mL. Since each insect could ingest (Mann Whitney, n.s.; Fig. 4B). For T. infestans, no differ- 20-30 mL of blood, the number of epimastigotes would ences in the number of parasites between EVs-exposed range from 2-3 x 10 . Control group was fed on blood nymphs and controls were observed (Mann Whitney, and blood + parasites with similar amounts of the treated n.s.; Fig. 5A-B). At 28 days p.i. the number of metacy- group in the respective days. The midgut and rectum of clis in the urine did not vary among controls and EV ex- a group of nymphs (n = 20) were individually dissected posed insects for both triatomine species (t test, p > 0.05, (Fig. 1) and homogenised in 30 μL of PBS (0.15 M NaCl Fig. 6A-B). All T. infestans nymphs released parasites in at 0.01 M sodium phosphate, pH 7.4) at 21-22 days post urine, but in numbers ~10 fold smaller than those found infection (p.i.). Quantification of parasites was made in in R. prolixus ones. Neubauer chamber. At 28 days p.i., a new feeding with Parasites are known release exosome-like EVs that citrated heat-inactivated rabbit blood was offered for the function as cell-to-cell effectors during the host-parasite (11,12,15,16,17) remaining nymphs (n = 15). Immediately after feeding, interaction. One of the initial studies showed the nymphs were transferred to 1.5 mL plastic tubes and that challenge of BALB/c mice with T. cruzi EVs exacer- (11) the urine produced during diuresis was collected to eval- bated parasite load, heart inf lammation and mortality. uate the percentage of metacyclic forms. 10 µL of each Later, it was demonstrated that T. cruzi could modulated sample were fixed in 10% methanol and stained with Gi- not only the innate but also the acquired immune events emsa®. The numbers of parasites were quantified by an- by activating TLR2, triggering cytokine production, (12,18) MAPKs activation and invasion. It is important to mention that the concentration of vesicles used in those studies ranged from 1-10 µg/mL. Here, pre-feeding with EVs was approximately 5 µg/mL. Although it may be not physiological, this concentration is within a range known to functionally activate vertebrate cells. Howev- er, reports regarding the role of EVs with respect to their invertebrate hosts are unknown. R. prolixus and T. infestans are the most used tri- atomine models due to due to existence of laboratory colonies. Both models were successfully used in our Fig. 1: basic diagrammatic representation of a digestive tract from a procedures and produced the expected infection pattern (14,15,16,17,18,19) triatomine bug. as previously reported. However, the abil- | Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 114, 2019 3 5 Fig. 2: extracellular vesicles (EVs) of Trypanosoma cruzi. (A) Scanning electron microscopy (SEM) of T. cruzi membrane shedding (bars: 1-5 µm), Magnification 32,657 x and (B) Scanning electron microscopy (SEM) of T. cruzi membrane shedding (bars: 1-5 µm) Magnification 80,000 x. Nanoparticle tracking analysis (NTA) (C) and (D) of T. cruzi EVs. Fig. 3: extracellular vesicles (EVs) of Trypanosoma cruzi. Transmis- sion electron microscopy (TEM) of T. cruzi membrane shedding (A) (lower magnification, bar: 200 nm) and (B) (higher magnification, bar: 100 nm). ity of T. infestans to take a blood meal in the artificial system was much lower than that of R. prolixus. Pre- feeding with EVs delayed the early migration of T. cruzi parasites to the rectum (21-22 days p.i.) in R. prolixus. Fig. 4: parasites found throughout the digestive tract of Rhodnius pro- This effect was not observed after 49-50 days p.i., sug- lixus. Dissections at 21-22 (A) and 49-50 (B) days after the infective gesting a transient effect of the EVs during the initial feeding. Each dot represents the quantification of parasites from one days of infection. However, this effect was not detected individual nymph and each horizontal bar corresponds to the median in T. infestans in both periods. The number of metacy- of the group evaluated. clics in EV exposed and controls did not vary for both vectors reinforcing their role only in the initial events of infection. Interestingly, the number of metacyclic try- belongs to the tribe Rhodniini, whereas T. infestans is (20) pomastigotes recovered in the urine of R. prolixus was from the Triatomini tribe. We thus believe that such 10-fold higher than in T. infestans. This result was very differences may be attributed to the species rather than surprising since Bug strain was originally isolated from the amount of ingested blood. Although in our model, T. infestans in Brazil, whereas the population of R. pro- pre-feeding with EVs affected early parasite migration lixus used in this study was from Honduras. R. prolixus only in R. prolixus, their role in transmission was not as- | 4 5 Larissa F Paranaiba et al. Fig. 6: metacyclic trypomastigotes found in the urine of Rhodnius pro- lixus (A) and Triatoma infestans (B) at 28 days p.i. Each dot represents the quantification of parasites in the urine sample of one nymph and Fig. 5: parasites found throughout the digestive tract of Triatoma in- each horizontal bar corresponds to the mean of the group evaluated. festans. Dissections at 21-22 (A) e 49-50 (B) days after the infective feeding. Each dot represents the quantification of parasites from one individual nymph and each horizontal bar corresponds to the median of the group evaluated. 5. Alves MJ, Colli W. Trypanosoma cruzi: adhesion to the host cell and intracellular survival. IUBMB Life. 2007; 59: 274-9. 6. Nogueira NF, González MS, Gomes JE, de Souza W, Garcia ES, sessed. Since the number of total parasites and metacy- Azambuja P, et al. Trypanosoma cruzi: involvement of glycoinosi- clics in later days of infection did not vary, it is not likely tolphospholipids in the attachment to the luminal midgut surface that EVs will affect transmission in triatomines. This is of Rhodnius prolixus. Exp Parasitol. 2007; 116(2): 120-28. different from L. major, where EVs were important for (10) 7. Soares RP, Torrecilhas AC, Assis RR, Rocha MN, Moura e Castro parasite transmission during the sand f ly bite. Despite FA, Freitas GF, et al. Intraspecies variation in Trypanosoma cruzi the presence of EVs, we showed that both vectors were GPI-mucins: biological activities and differential expression of able to develop metacyclics. However, in nature those α-galactosyl residues. Am J Trop Med Hyg. 2012; 87(1): 87-96. parasites are released in the feces and urine and are not 8. Szempruch AJ, Dennison L, Kieft R, Harrington JM, Hajduk SL. inoculated as in Leishmania. Sending a message: extracellular vesicles of pathogenic protozoan In vertebrate cells, it is already known that fusion parasites. Nat Rev Microbiol. 2016. 14(11): 669-75. of EVs is an important mechanism that promotes para- (11,18) 9. Campos JH, Soares RP, Ribeiro K, Andrade AC, Batista WL, site internalisation. Our results suggest that T. cruzi Torrecilhas AC. Extracellular vesicles: role in inflammatory re- EVs could be fusing to the epithelium of the midgut sponses and potential uses in vaccination in cancer and infectious and somehow promoting early parasite retainment in R. diseases. J Immunol Res. 2015; 2015: 832057. prolixus. This effect was transient and did not affect the 10. Atayde VD, Aslan H, Townsend S, Hassani K, Kamhawi S, Ol- number of metacyclics in both vectors. ivier M. Exosome secretion by the parasitic protozoan Leishmania AUTHORS’ CONTRIBUTION within the sand f ly midgut. Cell Rep. 2015; 13(5): 957-67. RPS, AAG, ACT and MNM conceived and planned the 11. Torrecilhas ACT, Tonelli RR, Pavanelli WR, da Silva JS, Schum- experiments; LFP, AAG and ACT performed experiments and acher RI, de Souza W, et al. Trypanosoma cruzi: parasite shed analysed data. All authors wrote and corrected the manuscript. vesicles increase heart parasitism and generate an intense inf lam- matory response. Microbes Infect. 2009; 11(1): 29-39. REFERENCES 12. Nogueira PM, Ribeiro K, Silveira ACO, Campos JH, Martins-Fil- 1. Pérez-Molina JA, Molina I. Chagas disease. Lancet. 2018; ho OA, Bela SR, et al. Vesicles from different Trypanosoma cruzi 391(10115): 82-94. strains trigger differential innate and chronic immune responses. J Extracell Ves. 2015; 4: 28734. 2. Antinori S, Galimberti L, Bianco R, Grande R, Galli M, Corbel- lino M. Chagas disease in Europe: a review for the internist in the 13. Palace-Berl F, Pasqualoto KFM, Jorge SD, Zingales B, Zorzi RR, globalized world. Eur J Intern Med. 2017; 43: 6-15. Silva MN, et al. Designing and exploring active N′-[(5-nitrofuran- 3. Schofield CJ, Dias JC. 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Vesicles as soma cruzi strains associated with their distinct interaction with carriers of virulence factors in parasitic protozoan diseases. Mi- host cells. J Extracell Ves. 2018; 7(1): 1463779. crobes Infect. 2012; 14(15): 1465-74. 19. Castro DP, Moraes CS, González MS, Ratcliffe NA, Azambuja 16. Marcilla A, Martin-Jaular L, Trelis M, de Menezes-Neto A, Osuna P, Garcia ES. Trypanosoma cruzi immune response modulation A, Bernal D, et al. 2014. Extracellular vesicles in parasitic dis- decreases microbiota in Rhodnius prolixus gut and is crucial for eases. J Extracell Ves. 2014; 1: 1-15. parasite survival and development. PLoS One. 2012; 7(5): e36591. 17. Torró LMP, Moreira LR, Osuna A. Extracellular vesicles in Cha- 20. Lent H, Wygodzinsky PW. Revision of the triatominae (Hemip- gas disease: a new passenger for an old disease. Front Microbiol. tera, Reduviidae), and their significance as vectors of Chagas dis- 2018; 9: 1190. ease. Bull Am Mus Nat Hist. 1979; 163: 125-520.

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