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Expression of Fluorescent Genes in T.Cruzi and T. Rangeli

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Expression of Fluorescent Genes in T.Cruzi and T. Rangeli VECTOR/PATHOGEN/HOST INTERACTION,TRANSMISSION Expression of Fluorescent Genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): Its Application to Parasite-Vector Biology PALMIRA GUEVARA,1 MANUEL DIAS, AGUSTINA ROJAS,2 GLADYS CRISANTE,2 MARIA TERESA ABREU-BLANCO, EUFROZINA UMEZAWA,3 MARTIN VAZQUEZ,4 4 2 5 MARIANO LEVIN, NESTOR AN˜ EZ, AND JOSE LUIS RAMIREZ Instituto de Biologõ´a Experimental, Universidad Central de Venezuela, Apartado Postal 48162, Caracas 1041A, Venezuela J. Med. Entomol. 42(1): 48Ð56 (2005) ABSTRACT Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green ßuorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of ßuorescent proteins. Tagged ßagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its ßuorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites. KEY WORDS Trypanosoma cruzi, Trypanosoma rangeli, green ßuorescent protein, mixed infection Trypanosoma cruzi Chagas, 1909, causes ChagasÕ dis- zymes, and it has proved invaluable for the in vivo ease, a debilitating and often incurable ailment affect- visualization of cell processes (Southward and Surette ing nearly 20 million people in endemic areas of South 2002). However, there have been few reports on the and Central America. In some of these areas, another use of GFP for examining parasiteÐvector interactions protozoa, Trypanosoma rangeli Tejera, 1920, shares (Bingle et al. 2001, Guevara et al. 2001). In the present insect vectors and mammalian hosts with T. cruzi work, the T. cruzi cloning vector pTREX-n (Vazquez (DÕAlessandro-Bacigalupo and Saravia 1992). Unlike and Levin 1999) was used to express GFP and DsRed T. cruzi, T. rangeli has pathological effects on the insect in different T. cruzi and T. rangeli isolates. The result- vector (Tobie 1965, Watkins 1971, An˜ ez 1984), but it ing tagged parasites were used to examine the life is harmless to the mammalian host (DÕAlessandro- cycles of T. cruzi and T. rangeli in the vector Rhodnius Bacigalupo and Saravia 1992). T. rangeli is transmitted prolixus Stal, 1859. In addition, in vitro cultured kidney to vertebrates by the bite of triatomine bugs (Tobie monkey cells were used to observe the invasion and 1965, An˜ ez 1984), whereas T. cruzi transmission occurs intracellular multiplication of GFP-tagged T. cruzi. by fecal contamination. Mixed infections with both species of parasites in mammalian and triatomine hosts are not uncommon (Hoare 1972). Materials and Methods In laboratory experiments, the innocuous, strongly ßuorescent green ßuorescent protein (GFP) is an Parasites. Venezuelan isolates of T. cruzi (MHOM/ important tool for tagging cells. GFP expression does Ve/92/2-92-YBM and MHOM/Ve/91/1-91-JMP) and not require cofactors such as ATP or reduced coen- T. rangeli (MCAN/Ve/82/Dog-82, IRHO/Ve/98/ Triat-1, IRHO/Ve/98/Triat-2, and MMAC/Ve/98/ Mono) and a T. cruzi Brazilian reference strain (CL 1 E-mail: palmiragt@@hotmail.com. 2 Universidad de los Andes, Facultad de Ciencias, Departamento de strain clone-Brener) (Cano et al. 1995) were used. Biologõ´a, Me´rida, 5101, Venezuela. Parasites were grown in Liver Infusion Tryptose 3 Instituto de Medicina Tropical de Sao Paulo, Universidad Sao (LIT) medium until they reached a density of 5 ϫ 106 Paulo, Sao Paulo, Brazil. cells/ml. GFP- and DsRed-tagged T. cruzi and 4 Institute for Genetic Engineering and Molecular Biology, Buenos Aires, Argentina. T. rangeli were cultured in NNN media supplemented 5 Instituto de Estudios Avanzados-MCT, Caracas, Venezuela. with 500 ␮g/ml of geneticin G418 antibiotic. 0022-2585/05/0048Ð0056$04.00/0 ᭧ 2005 Entomological Society of America January 2005 GUEVARA ET AL.: EXPRESSION OF FLUORESCENT GENES IN Trypanosoma 49 T. cruzi Cell Infection Assays. Subconßuent cultures primers, reaction conditions and ampliÞcation param- of LLC-MK2 cells were infected with metacyclic eters described in the original publications. forms of T. cruzi isolate GFP-MHOM/Ve/92/2-92- T. cruzi rDNA Group Typing. For this purpose, we YBM. After 48Ð72 h, free parasites were washed away examined a “group-speciÞc” PCR fragment found in and the infected LLC-MK2 cells were maintained in the 24S subunit ribosomal gene, as described previ- 2% fetal calf serum-RPMI-1640, at 37ЊC in 5% CO2. ously by Souto and Zingales (1993). Trypomastigotes (TCT) were obtained from cell su- Parasite DNA Isolation. Cultured parasites were pernatants and used for subsequent infection of new harvested at a cell density of 5 ϫ 106 ßagellates per LLC-MK2 cultures. milliliter and lysed by incubation in 10 mM Tris-HCl, Triatomine Bugs. Nymphs (fourth and Þfth instar) pH 7.5, 100 mM NaCl, 0.5% SDS, 1 mM EDTA, fol- of R. prolixus were reared in closed colonies for use in lowed by digestion with proteinase K (2 ␮g/ml). DNA this work (An˜ ez and East 1984). was isolated by phenol:chloroform extraction, and to- Triatomine Experimental Infections. Cultured tal nucleic acids were recovered by ethanol precipi- ßagellates were collected by centrifugation at 4,000 ϫ tation. The DNA of T. rangeli strain San Agustin was g. The supernatants were discarded and the pellets generously donated by Dr. John Swindle (Infectious were resuspended in deÞbrinated rabbit blood to a Disease Research Institute, Seattle, WA). concentration of 5 ϫ 106 parasites per milliliter. This GFP and DsRed Plasmid Constructs. A HindIII/ mixture was then placed in an artiÞcial feeding system XhoI fragment derived from pGFP5(S65T) containing coupled to a circulating water bath adjusted to 37ЊC the mgfp5(S65T) gene version of GFP (Siemering (Garcia et al. 1984). Batches of 25 bugs each were et al. 1996) was cloned into pTREX-n digested with allowed to feed for 30 min. Engorged insects were kept HindIII/XhoI (Fig. 2A), resulting in plasmid at 25ЊC with 80% humidity and a photoperiod of 12:12 pTREXn-GFP5(S65T). The DsRed construct was (L:D) h. Systematic observations were performed at made by replacing the GFP gene in pTREXn-GFP5 zero hour, daily up to day 15, and every 7 d thereafter (S65T) with an EcoRI/Not fragment derived from until day 31. Hemolymph was sampled from the cut plasmid pDsRed1.1 (BD Biosciences Clontech, Palo end of one leg per infected bug, smeared on a glass Alto, CA). The Þnal construct was named pTREXn- slide, and examined by ßuorescent and light micros- DsRed1.1 (Fig. 2A). pBsCalB1/CUB01 constructs ex- pressing GFP were obtained by replacing the CUB copy. Other specimens were dissected at different gene with an XbaI fragment derived from pEGFP, times and parts of their digestive tracts were teased containing the EGFP version of GFP (BD Biosciences apart and placed on glass slides for observation. Flu- Clontech) (Fig. 4B). The resulting construct, orescence observations were performed on an Axio- pBs:CalB1/CUB01-EGFP, was converted to the scope ßuorescent microscope (Carl Zeiss, Jena, Ger- DsRed vector by replacement of the EGFP gene many) by using an excitation wavelength of 520 nm with an XmaI/NotI fragment derived from plasmid and observing with a Þlter with a range between 450 pDsRed1.1 (Fig. 4B). and 490 nm. In this way, a compromise emission wave- Parasite Transfections and Selection of T. cruzi and length was reached that allows the simultaneous ob- T. rangeli Stable Fluorescent Cell Lines In Vivo. servation of green and red. Photographs were taken The electroporation protocol used for T. cruzi and with a fully automated MC-80 camera (Carl Zeiss). T. rangeli was as described by Hariharan et al. (1993). Because parasites were not Þxed, their movement pro- Cultured ßagellates were grown to mid-log phase, duced blurred images. 7 harvested by centrifugation, and washed with LIT Fluorescence Level Determination. A total of 10 media minus hemin and serum (LIT-HS). Cells were GFP-labeled parasites were adjusted to 500 ␮l in saline adjusted with LIT-HS to a Þnal concentration of 8.5 ϫ solution (0.85% NaCl), placed in quartz cuvettes, and 108/ml, 200 ␮g of plasmid DNA was added to 0.35 ml analyzed in a ßuorescence spectrophotometer (model of cell suspension in a 2-mm gap electroporation cu- F 2000, Hitachi, Tokyo, Japan) with excitation at 495 vette (BTX), and the mixtures were incubated at 4ЊC nm and detection at 515 nm. The background was set for 10 min. Cells were transfected by a single electric in comparison to the same concentration of nonßuo- pulse of 300 V, 1000 ␮F, and 100 ⍀ by using a Gene rescent cells, and ßuorescence was expressed as arbi- Pulser II (Bio-Rad, Hercules, CA). Electroporated trary units. cells were resuspended in 10 ml of complete LIT Typing of T. cruzi and T. rangeli Isolates. Before medium and incubated at 28ЊC. Forty-eight hours experimental infections, T. cruzi and T. rangeli isolates later, 500 ␮g/ml of G418 was added to the media. After were typed. For typing T. cruzi, we used two species- 15 d, the antibiotic was withdrawn. speciÞc polymerase chain reaction (PCR) assays: the Chromosomal Band Analysis. Pulse Þeld gel elec- Þrst targeted repeated sequences of the intergenic trophoresis (PFGE) of transformedT.
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