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Tsetse Immune Responses and Trypanosome Transmission: Implications for the Development of Tsetse-Based Strategies to Reduce Trypanosomiasis

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Tsetse Immune Responses and Trypanosome Transmission: Implications for the Development of Tsetse-Based Strategies to Reduce Trypanosomiasis Tsetse immune responses and trypanosome transmission: Implications for the development of tsetse-based strategies to reduce trypanosomiasis Zhengrong Hao*, Irene Kasumba*, Michael J. Lehane†, Wendy C. Gibson‡, Johnny Kwon*, and Serap Aksoy*§ *Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, New Haven, CT 06510; †School of Biological Sciences, University of Wales, Bangor LL57 2UW, United Kingdom; and ‡School of Biological Sciences, University of Bristol, Bristol BS8 IUG, United Kingdom Edited by John H. Law, University of Arizona, Tucson, AZ, and approved August 14, 2001 (received for review July 16, 2001) Tsetse flies are the medically and agriculturally important vectors Tsetse flies are in general refractory to parasite transmission, of African trypanosomes. Information on the molecular and bio- although little is known about the molecular basis for refracto- chemical nature of the tsetse͞trypanosome interaction is lacking. riness. In laboratory infections, transmission rates vary between Here we describe three antimicrobial peptide genes, attacin, de- 1 and 20%, depending on the fly species and parasite strain fensin, and diptericin, from tsetse fat body tissue obtained by (8–10), whereas in the field, infection with T. brucei spp. complex subtractive cloning after immune stimulation with Escherichia coli trypanosomes is typically detected in less than 1–5% of the fly and trypanosomes. Differential regulation of these genes shows population (11–13). Many factors, including lectin levels in the the tsetse immune system can discriminate not only between gut at the time of parasite uptake, fly species, sex, age, and molecular signals specific for bacteria and trypanosome infections symbiotic associations in the tsetse fly, apparently play a part in but also between different life stages of trypanosomes. The determining the success or failure of parasite infections (14). presence of trypanosomes either in the hemolymph or in the gut Tsetse flies have been shown to possess midgut lectin(s) that are early in the infection process does not induce transcription of capable of killing trypanosomes in vivo by a process resembling attacin and defensin significantly. After parasite establishment in programmed cell death (14), and there is also indirect evidence the gut, however, both antimicrobial genes are expressed at high to suggest that trypanosomes may be killed by an innate immune levels in the fat body, apparently not affecting the viability of response in the fly (15, 16). Although there is some information parasites in the midgut. Unlike other insect immune systems, the demonstrating antimicrobial activity (17–19), the prophenoloxi- antimicrobial peptide gene diptericin is constitutively expressed in dase cascade (20), lectin (21), and hemocyte types (22), no both fat body and gut tissue of normal and immune stimulated information is available on tsetse innate defense mechanisms at flies, possibly reflecting tsetse immune responses to the multiple molecular and biochemical levels. In addition to transmitting Gram-negative symbionts it naturally harbors. When flies were trypanosomes, tsetse flies also harbor multiple symbionts and immune stimulated with bacteria before receiving a trypanosome rely on these associations for nutrition and fecundity (23). The containing bloodmeal, their ability to establish infections was two gut symbionts, Sodalis glossinidius and Wigglesworthia severely blocked, indicating that up-regulation of some immune glossinidia, are Gram-negative bacteria closely related to Esch- responsive genes early in infection can act to block parasite erichia coli. How the trypanosomes and multiple symbionts can transmission. The results are discussed in relation to transgenic evade the natural immune mechanisms of tsetse is at present approaches proposed for modulating vector competence in tsetse. unknown. This is the first report, to our knowledge, on the molecular Glossina ͉ insect immunity ͉ vector control ͉ transgenesis ͉ symbiosis characterization of immune responsive genes from tsetse. By using a suppression subtractive hybridization approach, cDNA he life cycle of the parasitic African trypanosomes (Eugleno- fragments from Glossina morsitans morsitans were isolated from Tzoa: Kinetoplastida) in their insect vector, the tsetse fly fat body after immune challenge with E. coli and procyclic (Diptera: Glossinidae), begins when it feeds from an infected trypanosomes. We present data on the expression profiles of the mammalian host. For successful transmission, the parasite un- three antimicrobial peptide genes selected in this analysis, dergoes two stages of differentiation in the fly: first, establish- defensin, attacin, and diptericin, after challenge with bacteria and ment in midgut and then maturation in the mouthparts or trypanosomes by both microinjection and direct feeding ap- salivary glands. In the midgut, the mammalian bloodstream proaches. The regulation of immune peptide gene transcription parasites rapidly differentiate to procyclic forms and begin to in fat body was also studied in flies, which had established replicate (establishment). Once established in the midgut, try- parasite infections in the gut. In addition, the ability of tsetse panosomes migrate forwards to the proventriculus and the immune mechanism(s) to interfere with the establishment of mouthparts, where they begin to differentiate into epimastigotes parasite gut infections was investigated by stimulating tsetse and eventually colonize the proboscis or salivary glands, de- pending on the parasite species (1). Here they differentiate into metacyclic forms infective to mammals (maturation) and can be This paper was submitted directly (Track II) to the PNAS office. transmitted to the next host during blood feeding by the fly (2). Abbreviation: LPS, lipopolysaccharide. It is generally thought that during normal development in the fly, Data deposition: The sequences reported in this paper have been deposited in the GenBank there are no intracellular stages, although reports of intracellular database (accession nos. AF368906, AF368907, and AF368909). Trypanosoma brucei rhodesiense (3, 4) and Trypanosoma congo- §To whom reprint requests should be addressed at: Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, 60 College Street, lense (5) in the anterior midgut cells have been published. It is 606 LEPH, New Haven, CT 06510. E-mail: [email protected]. also thought that during normal infection, trypanosomes do not The publication costs of this article were defrayed in part by page charge payment. This cross an epithelial barrier to enter the fly, although there are article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. several reports of trypanosomes in the hemolymph of flies (6, 7). §1734 solely to indicate this fact. 12648–12653 ͉ PNAS ͉ October 23, 2001 ͉ vol. 98 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.221363798 Downloaded by guest on September 30, 2021 immune response(s) before delivering an infectious bloodmeal. Expression Analysis in Response to Immune Challenge. Three groups The potential to modulate the vector competence via transgen- of 40 flies each were microinjected with either 2 ␮l of PBS or live ϫ 5 esis is discussed in light of the role tsetse innate immune E. coli K12 XL-1 blue cells in PBS (OD600 0.6) or 1 10 responses play in trypanosome establishment. procyclic trypanosomes, respectively. Ten flies from each group were dissected at 6, 18, 30, and 48 h after microinjection, Materials and Methods respectively, and fat body was collected. Fat body was also Tsetse. The G. m. morsitans colony at Bristol University was collected from a control (unstimulated) group of flies 12 h after originally established from puparia collected in Zimbabwe. The their last bloodmeal. Similarly, three groups of 1-week-old flies ϫ 5 ͞ colony maintained in the insectary at Yale University was received bloodmeals containing 1 10 cells ml of cryopre- established from puparia obtained in 1994 from the Bristol served bloodstream or procyclic trypanosomes or E. coli, re- Tsetse Research Laboratory. Flies were maintained at 24 Ϯ 1°C spectively. Fat body was dissected and pooled from six flies in with 55–60% relative humidity and received defibrinated bovine each group 8 and 24 h after the bloodmeals were administered. Fat body from control (unstimulated) flies was similarly analyzed blood at Yale and horse and pig blood at Bristol every other day ␮ by using an artificial membrane system (24). 8 and 24 h after a regular bloodmeal. Total RNA (20 g per lane) was electrophoresed on 2% formaldehyde agarose gels and Immune Stimulation. Adult nonteneral G. m. morsitans (4–6 days transferred to nylon membranes (Hybond, Amersham Pharma- cia). The attacin and defensin specific hybridization probes were old) were microinjected through the wing base area of the prepared as described, and an RNA probe was generated for pteropleuron with 2 ␮l of a mixture of live E. coli K12 RM148 diptericin by using the RNA Labeling Kit (Amersham Pharma- at OD 0.4 in PBS containing 1 ϫ 104 procyclics of T. 600 cia, catalogue no. RPN 3100). Hybridization conditions were as congolense and T. brucei. For expression analysis, T. b. rho- described. The findings were confirmed by three separate desiense (strain Ytat 1.1) cells were used for immune challenge. experiments. cDNA Subtraction and Construction of Enriched Libraries. The In Vitro
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