Inducible suicide vector systems for Trypanosoma cruzi

Microbes and Infection 17 (2015) 440e450 www.elsevier.com/locate/micinf

Original article

Inducible suicide vector systems for Trypanosoma cruzi Yanfen Ma a, Louis M. Weiss a,b, Huan Huang a,* a b

Department of Pathology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Bronx, NY 10461, USA Department of Medicine, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 2 March 2015; accepted 8 April 2015 Available online 18 April 2015

Abstract Chagas disease caused by Trypanosoma cruzi is a major neglected tropical parasitic disease. The pathogenesis of this infection remains disputable. There is no suitable vaccine for the prevention. Attenuated live vaccines can provide strong protection against infection; however, there are the concerns about latent infection or reversion to virulence in such attenuated strains. A method to induce T. cruzi death would provide a critical tool for research into the pathophysiological mechanisms and provide a novel design of safe live attenuated vaccines. We established effective inducible systems for T. cruzi employing the degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR). The DHFR degradation domain (DDD) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins. T. cruzi lines with Alpha-toxin, Cecropin A and GFP under the control of DDD with a hemagglutinin tag (HA) were developed. Interestingly, amastigotes bearing GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all resulted in inducible cell death with these fusions, indicating that DDDHA protein is also detrimental to amastigotes. Furthermore, these strains were attenuated in mouse experiments producing no pathological changes and inoculation with these DDDHA strains in mice provided strong protection against lethal wild type infection. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Trypanosoma cruzi; Chagas disease; Vaccine

1. Introduction Chagas disease is considered one of the major neglected tropical parasitic diseases by the World Health Organization (WHO). It is caused by the flagellate protozoan, Trypanosoma cruzi (T. cruzi). The disease is contracted primarily in the rural areas of Mexico, Central and South America. Approximately 8 million people are currently infected with T. cruzi. Up to 30% of chronically infected people develop cardiac alterations and up to 10% develop digestive, neurological or mixed alterations which may require specific treatment [1]. The mechanism of chagasic heart disease remains unclear. Parasite persistence [2], autoimmunity [3], and microvascular abnormalities [1,4] have been studied extensively as possible pathogenic mechanisms. Experimental studies suggest that

* Corresponding author. Tel.: þ1 718 430 2143; fax: þ1 718 430 8543. E-mail address: [email protected] (H. Huang).

alterations in cardiac gap junctions [5] may be etiologic in the pathogenesis of conduction abnormalities. However, major debates exist on which factor plays the key role in chronic cardiomyopathy, and this has not been resolved among researchers because parasite persistence occurs in experimental models and in infected patients. Current drugs cannot eliminate intracellular parasites. Therefore, to this end, it is not possible to dissect out the factors related to parasite persistence in order to define the precise disease mechanisms. The development of a transgenic T. cruzi bearing an inducible detrimental/toxin gene would allow this parasite to undergo a self-destructive process, resulting in the death of intracellular parasites and the elimination of tissue parasitism. This would allow research that could evaluate the role of parasite persistence to the development of auto-immunity and other processes involved in the pathogenesis of this infection. Currently, no suitable vaccine is available for protecting against T. cruzi infection, despite considerable research in this area. Dead parasites, surface proteins, virus vaccine and DNA

http://dx.doi.org/10.1016/j.micinf.2015.04.003 1286-4579/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Y. Ma et al. / Microbes and Infection 17 (2015) 440e450

vaccine were tested as experimental vaccines [6e9,12]. Although these experimental vaccines provide some protections against T. cruzi infection, as compared to live attenuated T. cruzi vaccines, these approaches do not provide a strong and long-lasting immunity against T. cruzi infection. In some infectious diseases component vaccines have been effective (e.g. pneumococcal disease), but to date this has not been true for T. cruzi infections. A general principle in vaccinology is that the more similar a vaccine to the natural disease results in the better protective immune response. The success of live-attenuated microorganisms as vaccines against other pathogens supports this principle [10,11]. The efficacy of these vaccines depends on the use of live naturally attenuated organisms and a truly self-limited infection is necessary to attain a strong and long lasting protection [13]. For example, recently a genetically attenuated Plasmodium was tested as a vaccine and was successful [14]. In T. cruzi, however, attenuated parasites still poses health risks, since it is has been impossible to eliminate the parasitism in the immunized hosts and attenuated parasites may revert to virulence particularly when immunized hosts develop immune compromise. To overcome this obstacle, we sought to develop a new method to prevent persistence, based on the introduction of an inducible detrimental/toxic gene into T. cruzi, which results in killing the organism upon the induction of the detrimental/toxic gene. Such an inducible strain could be useful as a vaccine to induce protective immunity. Once a protective immune response is present, any latent parasites could be eliminated by inducing the detrimental/toxic gene producing a “sterile” immunity. Thus, the technique could be used as a bio-safety device. To create inducible suicidal T. cruzi, we needed an effective regulatory expression system in which the protein from a detrimental/toxic gene would be degraded in the absence of induction agents, and the transgenic T. cruzi for this purpose can be produced without being killed by the detrimental/toxic proteins. Previously, we established an inducible system for T. cruzi by expressing a target protein as a fusion with the destabilization domain (ddFKBP) of the “rapamycin binding protein” [15]. In the absence of the synthetic ligand, Shield-1, proteins bearing this domain are rapidly degraded by the cytoplasmic proteosome. However, the stabilization of protein bearing this domain can be rapidly achieved by treatment with and reversible binding of Shield-1 to ddFKBP [16e19]. Rapid upregulation of protein levels can be readily achieved in T. cruzi. A modified pTREX vector was used to express 3flagddFKBP fusion proteins [15]. This Shield-1 inducible system has proven very useful in tissue culture, however, limitations of this system include the cost of Shield-1 and its pharmacokinetics which make it difficult to use in animals. Another degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme has been reported and used in mammalian systems [20]. The DHFR degradation domain (DDD) can be stabilized by inexpensive folate analogs such as trimethoprim (TMP) [20]. This technique has been used to regulate protein stability in the mammalian central nervous system [20] and to regulate a Plasmodium falciparum protein [21]. However, the DDD system has not previously

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been established in T. cruzi. If the DDD system could be used as an inducible expression method in T. cruzi, it would provide another option for an inducible expression in this organism that could be used both in tissue culture and in animal models. In this paper, we report the establishment of a DDD system for T. cruzi. The DDD system was used to create suicidal T. cruzi strains. Amastigotes bearing one of the inducible proteins of GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin ADDDHA or DDDHA can be induced to undergo a selfdestruction process inside the cytoplasm of host cells. These strains were found to be attenuated in mouse experiments and infection of mice with these strains provided significant protection against lethal infection with wild type strains. Potentially, these strains may be used as vaccine strains against T. cruzi infections and the DDD technique can serve as a biosafety device. The combination of this approach with other methods such as gene deletion should significantly improve the safety of an attenuated live T. cruzi vaccine. 2. Materials and methods 2.1. Cell culture T. cruzi epimastigotes (Tulahuen) was grown at 26  C in liver digest-neutralized, tryptose medium (LDNT), supplemented with 10% FCS (Life Technologies, Gaithersburg, MD). Trypomastigotes were obtained by growth in human foreskin fibroblast cultures. The transgenic strains of trypomastigotes were obtained from inoculation of metacyclic trypomastigotes in human foreskin fibroblast cultures. Human foreskin fibroblasts were cultured in Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum 100 units of both Penicillin and Streptomycin/ml and 2 mM LGlutamine (all four reagents are from Invitrogen, Norwalk, CT). Cells are maintained in an incubator with 37  C and 5% CO2. 2.2. Construction of inducible expression vectors To construct two inducible toxin expression vectors, pTREX-3flagddFKBP-Alpha-toxin and pTREX3flagddFKBP-Pseaudomonas, open reading frames (ORFs) of alpha toxin of Staphylococcus aureus (GenBank accession number X01645.1) [22] and Pseudomonas aeruginosa Exotoxin A (GenBank accession number K01397.1) [23] were synthesized by GenScript Corporation (Piscataway, NJ). PCR was performed to generate DNA fragments for subcloning (Table 1 primer pairs 1, 2, and 3). These DNA fragments have appropriate restriction sites for subcloning into multiple cloning sites of pTREX vector [24] and the 3flag-ddFKBP DNA fragment was amplified by PCR using PTREX3flagddFKBP-EYFP as a template. The 3flag-ddFKBP DNA fragment was inserted to the N-terminal ends of both toxins to obtain two inducible toxin constructs, pTREX-3flagddFKBPAlpha-toxin and pTREX-3flagddFKBP-Pseudomonas (Fig. 1, number 1 and 2). To establish the DDD inducible expression

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Table 1 Primers for PCR and cloning of the inducible constructs. Primers 1. Alpha-toxin

Forward

Reverse 0

EcoR1 5 -CCGGAATTCATGATGAAA ATG AAAACACG-30 2. Pseudomonas Exotoxin A EcoR1 50 -CCGGAATTCATGCACCTG ATACCCCATTG-30 3. 3flagddFKBP EcoR1 50 -GGAATTCATGGACTACAAAGACCATGACG-30 4. DDDHA Cla1 50 -CCATCGATATGATCAGTCTGATTGCGGCGTT-30 5. EYFP Cla1 50 -CCATCGATGTGAGCAAGGGCGAGGAGCT-30 6. GFP-DDDHA HindIII 50 -CCCAAGCTTATGAGTAAAGGAGAAGAACT-30 7. DDDHA (30 -overhhand) 50 -ATCAGTCTGATTGCGGCGTTAGCGG-30 8. Alpha-toxin 50 -GTCTTGTCTAGAATTATGATGAAAATGAAAACACG-30 (both ends with overhand) 9.Pseudomonas 50 -GTCTTGTCTAGAATTATGCACCTGATACCCCATTGGA-30 (both ends with overhand) 10. Cecropin A 50 -GTCTTGTCTAGAATTATGAAATGGAAGTTATTCAA-30 (both ends with overhand) 11. DDDHA þ ATG 50 -GTCTTGTCTAGAATTATGATCAGTCTGATTGCGGCGTT-30 (both ends overhand)

system in T. cruzi, we constructed four DDD inducible expression vectors, using PCR to generate DNAs with appropriate restriction sites for subcloning (Table 1 primer pairs 4, 5 and 6). To create pTREX-DDDHA-EYFP and pTREX-GFP-DDDHA as two reporter constructs (Fig. 1, number 3 and 4), PGDBvm–GFP-DHFR(DD)-HA fusion (plasmid, kind gift of Dr. Michael White) was used as a template for PCR amplifications of GFP-DDDHA and DDDHA DNA fragments. GFP-DDDHA was ligated into

Xho1 50 -CCGCTCGAGTTAATTTG TCATTTCTTCTT-30 HindIII 50 -CCCAAGCTTTTACTTCAGGTCCTCGCGCG-30 EcoR1 50 -GGAATTCTTCCGGTTTTAGAAGCTCCAC-30 Cla1 50 -CCATCGATAGCGTAATCTGGAACATCGT-30 Sal1 50 -ACGCGTCGACTTATCGAAGCTTGAGCTCGA-30 Xho1 50 -CCGCTCGAGTCAAGCGTAATCTGGAACAT-30 50 -TCGTAAATGGCTCGATCAAGCGTAATCTGGAACAT-30 50 -CGCAATCAGACTGATATTTGTCATTTCTTCTTTTT-30 50 -CGCAATCAGACTGATCTTCAGGTCCTCGCGCGG-30 50 -CGCAATCAGACTGATACCCTTAGCAATCTGTGTT-30 50 -TCGTAAATGGCTCGATCAAGCGTAATCTGGAACAT-30

pTREX as pTREX- GFP-DDDHA. PTREX-3flagddFKBPEYFP was used as a template to amplify EYFP fragment by PCR. Both EYFP and DDDHA were ligated into pTREX to obtain pTREX- DDDHA- EYFP. To construct three inducible toxin vectors, pTREX-Alpha-toxin-DDDHA, pTREX-Pseudomonas-DDDHA and pTREX-Cecropin A-DDDHA (Fig. 1, number 5, 6, 7) by In-Fusion-Cloning, PCR was performed to generate three toxin DNA fragments with 15 bases overhanging at both ends, 5' overhang with the pTREX vector while 30 overhang with the DDDHA fragment (Table 1, primer pairs 7, 8,9 and 10). The template of Cecropin A [25] was also synthesized by GenScript Corporation. In-Fusion-Cloning of toxin DNAs and DDDHA with pTREX vector were performed following the instructions from the user manual (Clontech, Moutain View CA). DDDHA was amplified by PCR as an open reading frame by primers (Table 1, primer pairs 11) and the DNA fragment was ligated into pTREX vector by inFusion Cloning to produce a pTREX-DDDHA (Fig. 1, number 8). DNA sequencing was performed to confirm that appropriate DNA fragments were in these vectors. 2.3. Transfection, cloning and sorting of T. cruzi

Fig. 1. Design of constructs for inducible expression in pTREX vector. Both degradation domains, ddFKBP and DDD, were used in the pTREX constructs to mediate degradation of toxins or fluorescent proteins. The schematic diagrams represent: (1) Alpha-toxin N-terminally tagged with 3flagddFKBP. (2) Pseudomonas Exotoxin A N-terminally tagged with 3flagddFKBP. (3) EYFP N-terminally tagged with DDDHA. (4) GFP C-terminally tagged with DDDHA. (5) Alpha-toxin C-terminally tagged with DDDHA. (6) Pseudomonas C-terminally tagged with DDDHA. (7) Cecropin A C-terminally tagged with DDDHA. (8) DDDHA expresses as an open reading frame.

Constructs were introduced into epimastigotes (Tulahuen) by electroporation as previously described [26]. Briefly, epimastigotes in the late logarithmic growth phase in LDNT broth were collected and washed. 375 ml of the parasite suspension (1.4  108 to 2.0  108 cells/ml) was incubated with 50 mg construct DNA and adjusted to 400 ml of the final volume. Electroporation was performed in a BTX disposable cuvette using an Electro cell manipulator (BTX Genetronics, Inc., San Diego, CA) with one pulse delivered to the parasites in a setting of 375 V, 25 U, and 50 mF. Subsequently, the transfected parasite suspension was diluted with 10 ml of LDNT medium and G418 was included in the LDNT medium at a final concentration of 100 mg/ml. G418 was maintained for more than 4 weeks until parasites transfected with no DNA were no longer viable.

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pTREX-Alpha-toxin-DDDHA strain was subjected to single-cell cloning. Drug-selected metacyclic trypomastigotes were used to infect fibroblast cultures and obtain tissue derived trypomastigotes. Trypomastigotes were deposited into a 96well plate with fibroblast cultures at a density of 1 cell/well in Dulbecco's Modified Eagle medium. Each population from an individual well was considered an individual clone and clones were subjected to LDNT culture with G418 as epimastigote cell lines. Fluorescent epimastigotes bearing pTREX-GFP-DDDHA induced by 60 nM TMP-lactate for 24 h were sorted by MoFloXDP Cell sorter (Beckman Coulter). The sorted parasites were then cultured for inoculation of mice. 2.4. Immunofluorescence analysis (IFA) IFA was performed as previously described [27]. Briefly, the epimastigotes of T. cruzi (Tulahuen) were washed and then adhered to poly-L-lysine coverslips. For the intracellular amastigotes, T. cruzi infected Human foreskin fibroblast monolayers on coverslips were also washed. The coverslips with either epimastigotes or the infected monolayers were then fixed with 4% paraformaldehyde, permeabilized for 5 min with PBS, 0.3% Triton X-100, blocked for 1 h with PBS, 3% bovine serum albumin, 1% fish gelatin, 5% goat serum, 50 mM NH4Cl, and then incubated with primary antibody, Anti-HA Tag monoclonal antibody (Millipore, Temecula, CA), at 1:50 for 1 h at 37  C, washed three times in PBS and then incubated with secondary antibody (goat anti-mouse IgG Fluoresceiin linked, 1:500) for 45 min at 37  C. Coverslips were then washed three times in PBS, incubated DAPI solution (5ug/ml, Molecular Probes). Subsequently, 200 ml Fluoromount-G solution (SouthernBiotech, Birmingham, AL) was placed on the slides. The slides were examined using a Fluorescence microscope. Pre-immune serum or a secondary antibody alone was used as negative control. 2.5. Immunoblotting Immunoblotting for T. cruzi lysates were described previously [27,28]. Briefly, samples of lysates from the T. cruzi (150 mg total protein per lane) were resolved on a 10% SDSpolyacrylamide gel and transferred onto nitrocellulose membrane, blocked with 5% non-fat milk and detected with antiHA Tag monoclonal antibody (1:1000 dilution) and secondary antibody conjugated with alkaline phosphatase (1:2000), and then visualized by using BCIP/NBT as substrate (Roche, Bavaria, Germany) using standard techniques. Anti-Alpha tubulin antibody (Sigma, St. Louis, MO) was used for loading control. 2.6. Detection of apoptosis The analysis of apoptotic events in intracellular parasites lodged within Human foreskin fibroblasts was carried out using TACS® 2 TdT-Blue Label in situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD), following the

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manufacturer's protocol. Briefly, coverslips with 80% confluent cells were infected with the transgenic T. cruzi or parental strain for 2e3 days. Afterwards, the culture medium supplemented with 250 nM TMP-lactate (Sigma, St. Louis, MO) was used to induce the expression of fusion proteins (e.g. GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA) for different time points. The culture medium without TMP-lactate was used for negative control. To fix the coverslips for analysis, media were removed from cells and the coverslips were rinsed once with PBS. Cells were fixed with 3.7% buffered formaldehyde for 20 min at room temperature and then washed with PBS. Afterwards, coverslips were mounted with 100 ul of Cytonin™ and stored at 4  C for analysis within 7 days. For apoptotic DNA labeling, coverslips were washed, permeabilized with 100 ul Proteinase K solution for 15 min at 37  C, treated with Quenching Solution (Methanol and 3% hydrogen peroxide) for 5 min, incubated with Labeling Reaction Mix containing TdT Enzyme and dNTPs for 60 min at 37  C and then terminated reactions by adding TdT Stop Buffer. Samples were further incubated with 50 ul of Strep-HRP Solution for 10 min at 37  C and then incubated with Blue Label Solution for 7 min. Coverslips were covered with Fluoromount-G solution and observed under light microscope. 2.7. Immunization with transgenic strains and challenge with lethal wildtype infection in mice To test whether the transgenic T. cruzi could provide immune protection in animal, we used 7-week old male C3H mice (Charles River Laboratories, Kingston, NY) which are highly susceptible to the Tulahuen strain. Transgenic Tulahuen strains respectively bearing a pTREX- Alpha-toxin-DDDHA (clone 3), pTREX-GFP-DDDHA (sorted fluorescent cells), pTREX- Cecropin A-DDDHA and pTREX-DDDHA were used to inoculate ten male C3H mice with 5000 tissue derived trypomastigotes per mouse. A week post-infection, 5 mice in each inoculated group were administrated with TMP-lactate in drinking water (30 mg/100 ml) until day 42 of postinoculation while another 5 mice were given normal water without TMP-lactate. As a control to see if TMP-lactate has an effect on the parental strain, 10 age matched C3H mice were infected with Tulahuen wildtype (5000 tissue derived trypomastigotes/mouse) and administrated with TMP-lactate in drinking water (30 mg/100 ml). At 42 days post-inoculation, all mice with pre-inoculation of transgenic strains were challenged with 4.6  105 tissue derived wildtype Tulahuen strain. 10 age and sex matched C3H mice without pre-inoculation of transgenic strains but only with mock injections were used as controls. Parasitemia of the infected mice was observed by light microscope. Survival rates of each group were recorded daily. Heart tissues from newly dead mice in control group (from day11e13 post-challenge) and from the sacrifice of preinoculated groups (on day 45 post-challenge) were fixed in 10% buffered formalin for 48 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Section and Hematoxylin and eosin stain (H&E) (at Albert Einstein

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facility) were performed on these hearts to observe the pathological changes. 3. Results 3.1. The ddFKBP system does not permit the development of inducible death T. cruzi strains We initially evaluated the ability of the ddFKBP inducible system to regulate a toxic protein in T. cruzi. Transfections with pTREX-3flagddFKBP-Alpha-toxin and pTREX3flagddFKBP-Pseudomonas-toxin resulted in cell death of epimastigotes, in the absence of Shield-1, despite of multiple attempts. This suggests that the ddFKBP system cannot mediate sufficient degradation of either toxin and the “leaky” phenotype resulted in the presence of toxic peptides inside the transformants which killed these organisms. Based on the Alpha-toxin literature [39], we subsequently, constructed two additional inducible vectors Alpha-toxin, pTREX3flagddFKBP-Alpha-toxin1-282 and pTREX-3flagddFKBPAlpha-toxin1-293 with a deletion in the C-terminal of the open reading frame. The deletion of the C-terminal end reduced the toxic effects of this toxin [39]. With the transfection of these two vectors, stably transfected epimastigotes were obtained; however, when Shield-1 (2 mM) was added to stabilize the 3flagddFKBP- partial Alpha-toxins, we did not observe any phenotypes in epimastigotes or amastigotes for either Alpha-toxin1-282 or Alpha-toxin1-293 (data not shown). 3.2. The DHFR degradation domain mediates inducible protein expression in epimastigotes of T. cruzi In order to establish another inducible expression system in T. cruzi, we tested the DDD system by constructing both Nterminal and C-terminal tags of DDD with a hemagglutinin tag (DDDHA) with the fluorescent proteins EYFP and GFP in a pTREX vector. These vectors are pTREX-DDDHA-EYFP (N terminal tag) and pTREX-GFP-DDDHA (C terminal tag). Both vectors were transfected into epimastigotes and stable transformants were obtained. The DDD system mediated degradation of these fluorescent proteins efficiently because no fluorescent epimastigotes were observed in the stable transformants bearing these vectors. Then, we tested whether TMP could stabilize the fusion proteins, DDDHA-EYFP or GFPDDDHA. TMP was first dissolved in DMSO as a 10 mM stock solution as the literature indicated [20]. Using the stock solution of TMP, we performed a dose dependent experiment in these transformants. Aliquots of the TMP stock solution in DMSO were added to the culture transformants bearing either DDDHA-EYFP or GFP-DDDHA to make the final concentration from 1 nM to 1 mM. For several days, no fluorescent parasite was observed under microscope and immunoblot with anti-GFP antibody using parasite lysates also failed to detect signals, indicating that TMP in DMSO as stock solution failed to stabilize the fusion proteins (data not shown). We then tested TMP-lactate because this compound is more soluble

and stable than TMP. We directly dissolved TMP-lactate in LDNT as 10 mM stock solution and then used this stock TMPlactate solution for further dilutions. By this method, at the concentration from 10 to 250 nM of TMP-lactate, fluorescent epimastigotes were easily observed in transformants bearing GFP-DDDHA (Fig. 2A). Immunoblot with parasite lysates from epimastigote bearing GFP-DDDHA induced with 10 nMe120 nM of TMP-lactate was performed. There was a dose dependent increase of stabilization of GFP-DDDHA (Fig. 2B), but TMP-lactate concentrations beyond 120 nM did not demonstrate any additional signal in immunoblot (data not shown). When 60 nM TMP-lactate was added to epimastigote culture medium, stabilization of GFP-DDDHA fusion protein was detected by immunoblot within 30 min and it reached the peak at 12 h in presence of this drug (Fig. 2C). However, both TMP and TMP-lactate did not stabilize the DDDHA-EYFP fusion protein (data not shown). Therefore, further experiments are required to investigate the stabilization of this N-terminal tag protein in this organism. 3.3. The DHFR degradation domain was sufficient to mediate degradation of Alpha-toxin and Cecropin A in epimastigotes To test whether the DDD domain could mediate the degradation of toxin genes effectively, we constructed three inducible C terminal DDDHA tagged toxin constructs, pTREX-Alpha-toxin-DDDHA, pTREX-Pseudomonas (Exotoxin A) -DDDHA and pTREX-Cecropin A-DDDHA. Transfection of pTREX-Pseudomonas-DDDHA resulted in cell death of all transfectants and no stable transformant could be obtained despite of multiple attempts, indicating that DDD probably cannot sufficiently degrade the Pseudomonas toxin resulting in cell death. Transfection of pTREX-Alpha-toxinDDDHA resulted in stable transformants. Addition of TMPlactate (250 nM) achieved a stabilization of the Alpha-toxinDDDHA in epimastigotes as the protein could be detected by both IFA and immunoblot with anti-HA mAb (Fig. 2D and E). However, in epimastigotes no obvious phenotype was seen with stabilization of Alpha-toxin-DDDHA. Transformants bearing pTREX-Cecropin A-DDDHA grew slowly in epimastigote culture and the expression of Cecropin A-DDDHA was at a low level. IFA and immunoblot using anti-HA mAb did not demonstrate stabilization of Cecropin A-DDDHA after addition of TMP-lactate in epimastigotes (data not shown). 3.4. Intracellular amastigotes died when GFP-DDDHA, DDDHA, Alpha-toxin-DDDHA and Cecropin ADDDHA were induced with TMP-lactate We tested whether the DDD system could be used to regulate inducible expression of proteins in intracellular amastigotes. The epimastigotes carrying pTREX-GFP-DDDHA were differentiated into trypomastigotes and then used to infect a fibroblast monolayer. Afterwards, TMP-lactate was added to the culture medium. GFP-DDDHA was clearly stabilized by TMP-lactate in intracellular amastigotes as many intracellular

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Fig. 2. The DHFR domain mediates inducible protein expressions in epimastigotes. (A) Fluorescence microscopy demonstrating epimastigotes bearing pTREXGFP-DDDHA become fluorescent in presence of 120 nM TMP-lactate for 48 h. Without TMP-lactate induction, the control cells are not fluorescent. Scale bar ¼ 10 mm. (B) Immunoblot demonstrating stabilization of GFP-DDDHA in epimastigotes increases with increase of TMP-lactate dose. The loading is150 mg parasite lysate protein per lane and Alpha-tubulin is used to verify the loading equivalency. (C) Immunoblot demonstrating the time cause of stabilization of GFPDDDHA in epimastigotes by 60 nM of TMP-lactate. Note that stabilization of GFP-DDDHA in epimastigotes is fast. It reaches the peak in 12 h. The loading is 150 mg parasite lysate protein per lane and Alpha-tubulin is used to verify the loading equivalency. (D) DHFR domain mediates inducible Alpha-toxin expression in epimastigotes. IFA demonstrating stabilization of Alpha-toxin-DDDHA in epimastigotes by 60 nM TMP-lactate. Without TMP-lactate induction, the control cells are not fluorescent by IFA. Scale bar ¼ 10 mm. (E) Immunoblot demonstrating a weak band of Alpha-toxin-DDDHA can be detected in parasite lysate from epimastigotes induced with 60 nM TMP-lactate for 24 h. The loading is150 mg parasite lysate protein per lane and Alpha-tubulin is used to verify the loading equivalency.

amastigotes were green. IFA also demonstrated that the GFPDDDHA protein was expressed in amastigotes (Fig. 3A). However, the intracellular amastigotes were then observed to undergo a process of self-destruction as many amastigotes lost normal morphology and then died after 7 days of 250 nM TMPlactate addition while the control amastigotes without TMPlactate and without stabilization of GFP-DDDHA grew normally (Fig. 3B). After 14 days of TMP-lactate induction, intracellular amastigotes were not observable in the monolayer by microscope. TMP-lactate by itself had no killing effect on the parental wildtype amastigotes (Tulahuen strain) and the monolayer was destroyed within 7days by the parasites in presence of 250 nM TMP-lactate (Fig. 3C). In addition, stabilization of DDDHA alone by TMP-lactate also killed intracellular amastigotes (Fig. 3D and E). Epimastigotes bearing pTREX-Alpha-toxin-DDDHA were differentiated into metacyclic trypomastigotes and used to infect Human foreskin fibroblasts. The addition of TMPlactate was able to induce stabilization of the Alpha-toxinDDDHA fusion protein. IFA demonstrated expression of the Alpha toxin-DDDHA protein inside intracellular amastigotes within 24 h induction and the fusion protein was then targeted to the amastigote membrane with 96 h of TMP-lactate induction (Fig. 3F and G). Targeting was not seen with GFPDDDHA or with DDDHA alone confirming that the Alphatoxin peptide was responsible for targeting. The induction of Alpha-toxin-DDDHA protein resulted in massive selfdestruction of amastigotes. Very large degenerating amastigotes were observed within 24 h after the addition of TMPlactate to the culture medium (Fig. 3H, middle panel). After

the addition of 250 nM TMP-lactate in culture medium for 7 days, amastigotes bearing pTREX-Alpha-toxin-DDDHA lost all normal morphology and were clearly dead (Fig. 3H, right panel). Amastigotes bearing pTREX-Alpha-toxin-DDDHA without the TMP-lactate induction grew normally during this 7 day period (Fig. 3H, left panel). Induction of Cecropin ADDDHA by TMP-lactate also resulted in T. cruzi intracellular cell death (Fig. 3I and J). To determine whether the intracellular cell death was due to apoptosis-like cell death, we used TACS® 2 TdT-Blue Label in situ Apoptosis Detection Kit, which is designed for the detection of apoptosis in cell culture. This method can detect DNA fragmentation in situ. After 48 h induction with TMPlactate, the majority of amastigotes expressing GFPDDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all stained positive by this method, indicating that apoptosis-like cell death of intracellular amastigotes occurred with the expression and stabilization of these fusion proteins (Fig. 4A, B, C and D). The majority of these transgenic amastigotes had no staining without the induction of the fusion proteins. The same treatment of TMP-lactate to the parental strain (Tulahuen amastigote) in fibroblast monolayer cultures did not result in any positive staining (Fig. 4E). 3.5. Transgenic strains are highly attenuated and provide protection against lethal infection with wild-type T. cruzi To investigate whether these transgenic T. cruzi strains can be used as potential animal or human vaccine strains, we

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Fig. 3. Expression of DDDHA fusions is detrimental to intracellular amastigotes and expression of toxin DDDHA fusions kills amastigotes. Four strains of transgenic T. cruzi are tested using TMP-lactate to stabilize the DDDHA fusion proteins; panels represent: A to C, GFP-DDDHA; D to E, DDDHA as open reading frame; F to H, Alpha-toxin-DDDHA and I to J, Cecropin A-DDDHA. (A) IFA demonstrating intracellular amastigotes with stabilized GFP-DDDHA protein by 250 nM TMP-lactate for 24 h. Without TMP-lactate induction, GFP-DDDHA is not detectable (control). Scale bar ¼ 10 mm. (B) Intracellular amastigotes gradually disappear by stabilization of GFP-DDDHA with 250 nM TMP-lactate for 7 days. Control amastigotes without TMP-lactate grow normally. Scale bar ¼ 20 mm. (C) Wildtype Tulahuen, both with or without 250 nM TMP-lactate (control), destroys Human foreskin fibroblast monolayers within 7 days, indicating that 250 nM TMP-lactate does not kill the intracellular amastigotes. Scale bar ¼ 20 mm. (D) IFA demonstrating stabilization of DDDHA protein by 250 nM TMPlactate for 24 h. Without TMP-lactate induction, DDDHA protein is not detectable (control). Scale bar ¼ 10 mm. (E) Expression of DDDHA results in amastigote death with 250 nM TMP-lactate for 7 days. Control amastigotes without TMP-lactate grow normally. Scale bar ¼ 20 mm. (F) IFA demonstrating intracellular amastigotes with stabilized Alpha-toxin-DDDHA protein by 250 nM TMP-lactate for 24 h, without TMP-lactate Alpha-toxin-DDDHA is not detectable (control). At this time; the toxin is located in cytoplasm of amastigotes. (G) IFA demonstrating more Alpha-toxin-DDDHA protein targets to membranes of amastigotes with the induction of TMP-lactate for 96 h (A larger image is included to have better view of membrane targeting). Without TMP-lactate induction, Alpha-toxinDDDHA is not detectable (control). Scale bar ¼ 10 mm. (H) Expression of Alpha-toxin-DDDHA kills the intracellular amastigotes. With 250 nM TMP-lactate for 24 h (middle panel), huge amastigotes appears, arrow heads are pointing to huge amastigotes. With 250 nM TMP-lactate for 7 days (right panel), amastigotes disappear from Human foreskin fibroblast monolayers. Without the induction of TMP-lactate, amastigotes grow normally (left panel). Scale bar ¼ 20 mm. (I) IFA demonstrating stabilization of Cecropin A-DDDHA by 250 nM TMP-lactate for 24 h, without TMP-lactate induction, Cecropin A-DDDHA is not detectable (control). Scale bar ¼ 10 mm. (J) Expression of Cecropin A-DDDHA kills the intracellular amastigotes. With 250 nM TMP-lactate for 7 days, amastigotes disappear from Human foreskin fibroblast monolayers. Without the induction of TMP-lactate, control amastigotes grow normally. Scale bar ¼ 20 mm.

inoculated groups of C3H mice with each strain of transgenic trypomastigotes (GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA respectively) at 5000 tissue derived trypomastigotes per mouse. These mice received TMP-lactate treatment (30 mg/100 ml drinking water) starting one week after the transgenic parasite inoculation. Parasitemia was not detectable (assessed by microscopy from day 7e42 following inoculation) and all infected mice survived appearing healthy during the acute infection course. Furthermore, another group of mice inoculated with these transgenic strains that were maintained without administration of TMP-lactate

also did not develop parasitemia and appeared healthy, suggesting that these strains are highly attenuated. To examine if TMP-lactate by itself could have an effect on T. cruzi infection in mice, sex and age matched C3H mice were infected with Tulahuen wildtype (5000 tissue derived trypomastigotes/ mouse) and given TMP-lactate in their drinking water (30 mg/ 100 ml); all of these wild type T. cruzi infected, TMP-lactate treated mice died within 21 day with high parasitemia (2.8  107/ml). This indicates that administrating TMP-lactate at this concentration in drinking water does not affect this infection by itself. All of the transgenic T. cruzi inoculated

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Fig. 4. Apoptosis-like cell deaths with stabilizations of DDDHA fusion proteins. Analysis of apoptotic events in intracellular parasites lodged within Human foreskin fibroblasts was carried out using TACS® 2 TdT-Blue Label in situ Apoptosis Detection Kit. After 48 h induction with TMP-lactate, the majority of amastigotes expressing GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all stained positive by this method. (A) GFP-DDDHA. (B) Alpha-toxin-DDDHA. (C) Cecropin A-DDDHA. (D) DDDHA. (E) Wildtype Tulahuen strain. Control: no TMP-lactate for induction. TMP-lactate 48 h: 250 nM TMP-lactate in culture medium for 48 h to stabilize the fusion proteins. Scale bar ¼ 20 mm.

mice were challenged with a lethal infection (4.6  105 tissue derived trypomastigotes, wildtype Tulahuen strain) and all of them survived and looked healthy. These mice did not develop parasitemia even with this heavy infection. Control (i.e. no transgenic pre-inoculation) C3H mice that were sex and age matched developed a high parasitemia and then died by day 13 (Fig. 5A and B). Histological examination demonstrated that there were no pathological changes or amastigotes in the hearts of the pre-inoculated mice challenged with a lethal wild-type infection, but the control mice had a high parasitic burden in their hearts and severe myocarditis (Fig. 5C). 4. Discussion An inducible self-destructive T. cruzi strain will be valuable for study of the pathogenesis of Chagas disease as it will allow examination of factors related to parasite persistence and how these contribute to the development of Chagas disease. The strains we have developed allow regulated cell death in vitro and can be used to examine parasite specific effects on host cells once the parasite is killed. Furthermore, these strains can be used to immunize host species to achieve sterile immunity. Therefore, this method may ease the concerns of health risk such as latent infection of a live attenuated T. cruzi vaccine. Thus, the inducible detrimental/toxic proteins can be used as a bio-safety device in attenuated live vaccine development and should eliminate intracellular infection in animals.

An effective inducible system is key to the success of making a self-destructive T. cruzi. Although a tetracyclineinducible expression system is established [29], this inducible system is not suitable for the study of intracellular life stages since the T7 polymerase and tetR genes are expressed from an episome. The pTREX ddFKBP vector allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages [15]. However, transfections of both constructs, pTREX-3flagDDAlpha-toxin and pTREX-3flagDD-Psudonomas, were lethal to epimastigotes, indicating that the ddFKBP system could not mediate sufficient degradation of Alpha-toxin and Pseudomonas. The DDD system can be stabilized by inexpensive folate analogs such as trimethoprim (TMP) in Plasmodium and human [20]. The DDD system with C-terminal fusion of GFP degraded the fusion protein efficiently in absence of induction. A folate analog TMP-lactate was able to induce stabilization of GFP-DDDHA in a dose and a time dependent manner in T. cruzi. The DDD system is effective in degrading both Alphatoxin and Cecropin A and cell lines bearing both toxin fusion proteins in T. cruzi were obtained. Interestingly, induction of Alpha-toxin-DDDHA and Cecropin A-DDDHA stabilization with TMP-lactate in epimastigotes did not result in obvious cell death. This may be due to the efficient degradation and low level stabilization of both toxin proteins in epimastigotes. We unexpectedly made a discovery when we tested whether the DDD system could be regulated to stabilize fusion proteins

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Fig. 5. The transgenic parasites are highly attenuated and inoculation with these strains in mice produces protection against lethal challenge infection. (A) With a lethal dose of the wild-type Tulahuen, parasitemia is not detectable in mice pre-inoculated with any of the transgenic DDDHA T. cruzi strains (Tulahuen strain bearing one of the fusion proteins: GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA or DDDHA). Non-pre-inoculated mice developed high parasitemia and died. (B) All mice immunized with a transgenic DDDHA T. cruzi strain survived infection while non-immunized mice died within 13 days of the lethal challenge infection (n ¼ 10). (C) Immunization protects heart from infection and damage. Shown are H&E staining of heart tissues. Note that Non-pre-inoculation mice (control mice died at day 13 of post-lethal infection) have heavy infection in the hearts with a lot of amastigotes inside the myocardium and the hearts develop severe myocarditis. Arrow head indicates a big nest of intracellular amastigotes. In pre-inoculated mice with transgenic strains bearing one inducible proteins, DDDHA, GFP-DDDHA, Alpha-toxin-DDDHA or Cecropin A-DDDHA, there are no amastigotes and inflammation (scarified at day 45 of post-challenge). Light microscope with 20 magnification. Scale bar ¼ 25 mm.

in intracellular amastigotes. Human foreskin fibroblasts were infected with pTREX-GFP-DDDHA strain and in absence of induction, amastigotes were replicating inside the host cells normally. Then, 250 nM TMP-lactate was added to the culture medium to induce GFP-DDDHA stabilization. After the induction with TMP-lactate for seven days the majority of intracellular amastigotes died while the amastigotes without TMP-lactate grew normally. Since expression of GFP has been used previously in T. cruzi and GFP does not show any toxic effect on T. cruzi [30], we believe it is the DDDHA peptide that is detrimental to this organism when it is stabilized and accumulates in amastigotes. In T. cruzi, DHFR-TS is a single copy gene which codes for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase [31,32]. This enzyme catalyzes sequential reactions in the biosynthesis of Thymidine monophosphate. Therefore inhibition of this enzyme results in thymidine synthesis defect and cell death. We hypothesize that the stabilization of the bacterial DDD exerts a dominant negative effect on this organism which inhibits the function of the endogenous T. cruzi DHFR-TS pathway. To test this hypothesis, we made a construct to express DDDHA alone. Amastigotes bearing pTREX-DDDHA were killed by the stabilization of DDDHA with TMP-

lactate, confirming that accumulation of DDDHA is detrimental to amastigotes. Thus, the bacterial DDD system by itself, without toxins, can be used as a tool for inducible killing of T. cruzi intracellular form. TMP-lactate alone does not kill the parasites, as 250 nM TMP-lactate treated parental amastigotes grew normally and eventually destroyed the infected fibroblast monolayers. The toxin strains are expected to be killed not only by the DDDHA peptide but also by the stabilization of the toxins they express. Alpha-toxin has been shown to cause damage by forming heptameric pores in cell membranes. This pore allows the exchange of monovalent ions, resulting in DNA fragmentation and eventually apoptosis [33]. At high concentrations the toxin absorbs nonspecifically to the lipid bilayer forming large, Ca2þ permissive pores resulting in massive necrosis [33]. TMP-lactate induced stabilization of the Alpha-toxinDDDHA fusion resulted in massive destruction of amastigotes. Some cell death could be observed by light microscopy within 24 h. IFA demonstrated that the majority of the stabilized Alpha-toxin-DDDHA protein was eventually targeted to amastigote membrane, which can destroy the integrity of the membrane and kill the parasites. Alpha-toxin forms pores in host cell membrane when it is secreted by the bacteria. In our

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system, the toxin is expressed inside the organism and the fusion toxin is degraded in absence of TMP-lactate. When it is stabilized by TMP-lactate, the toxin starts to kill the parasites by binding to membranes. It is not secreted from the parasites and even when if lysis of the parasite was to occur there should be limited amounts of free toxin. We, therefore, believe that this toxin would not affect the host cells. To this end, we have only seen binding of the fusion target to T. cruzi membranes and has never observed any fusion toxin appearing in host cells using IFA employing anti-HA mAb (which picks up the HA tag in the fusion protein). Furthermore, we do not see any toxic effects in monolayers or in mice. Cecropins are antimicrobial peptides [34]. Cecropins lyse bacterial cell membranes; they also inhibit proline uptake and cause leaky membranes. Cecropin A toxin has been used for the killing of epimastigotes in triatomine vectors by a symbiotic bacterium, Rhodococcus rhodnii [35,36]. Epimastigote transformants bearing pTREX-Cecropin A-DDDHA grow slowly. Accumulation of Cecropin A-DDDHA in amastigotes resulted in cell death. Therefore, the toxin strains possess two mechanisms of inducible killing. The accumulation of DDDHA is detrimental and the stabilization of toxins is toxic to the organism. To determine whether the intracellular cell death could be due to apoptosis-like cell death, we used a method that can detect DNA fragmentation in situ. After 48 h induction with TMP-lactate the majority of amastigotes expressing these fusion proteins stained positive, indicating that apoptosis-like cell death in amastigotes occurred with the expression of all these proteins. It has been reported that T. cruzi could undergo apoptosis-like cell death [37,38]. However, the pathways leading to apoptosis-like cell death in this organism require further explorations. In mouse experiments, C3H mice were used for the experiments because these mice are highly susceptible to the Tulahuen strain with infection being lethal within 21 days. All four transgenic Tulahuen strains, each bearing one of the DDD fusions, were tested in male C3H mice that were administrated TMP-lactate in drinking water starting a week after infection to induce self-destruction of the parasites. In all cases parasitemia was not detectable. All of these mice survived and did not develop parasitemia nor cardiac pathology with a lethal challenge by a wildtype strain. The data indicate that a strong immune protection is obtained by the pre-inoculations with the transgenic strains. The immune profile in mice in response to the inoculation of these transgenic T. cruzi should help us understand immune protection against lethal infection. A detailed analysis for the immune profile with inoculation of these transgenic strains is planned as future studies by our laboratory group. Mice inoculated with these four types of transgenic T. cruzi that were only given water (i.e. no TMP-lactate) also survived and did not develop parasitemia. This indicates that with these transgenic genes (DDDHA, GFP-DDDHA, Alpha-toxinDDDHA and Cecropin A-DDDHA), parasites are highly attenuated in C3H mice. It is possible that partially degraded gene products inside T. cruzi still interfere with T. cruzi

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infectivity and its ability to evade immune responses from the host. Thus, the virulence of the parasites has been reduced in vivo. DDDHA or DDDHA fusion proteins have been integrated to the T. cruzi genome. These strains can be maintained with G418 selection. For vaccination with these strains, there will be a short period without selection of G418. Strains with multiple integrations of DDDHA or DDDHA fusion proteins in the genome can increase the expression of DDDHA or DDDHA fusion proteins and enhance sensitivity to TMPlactate. Thus, this approach can reduce the dosage of TMPlactate to avoid its side effects. In summary, this paper reports a new inducible system to regulate gene products in T. cruzi. The bacterial DDD system can be used to create inducible suicidal T. cruzi amastigotes. T. cruzi strains bearing this system are highly attenuated and can provide strong protection against lethal infection. This system has significant potential as a bio-safety device in T. cruzi which can be used in combination with other techniques such as gene deletion or radiation to develop safe animal and human vaccines. These DDD strains can also be used for in vitro pathogenesis studies, to examine cell changes that persist after infection when the parasite is eliminated from its host cells. To create a strain to study pathogenesis in vivo, a similar approach can be applied, but we need to make a strain that is not as highly attenuated in animal infection. We plan to investigate other systems in order to create such strains for disease mechanism research in vivo. Conflict of interest There is no conflict of interest. Acknowledgments We thank Dr. Jinghang Zhang at Flow Cytometry Facility of Albert Einstein College of Medicine for the assistance. This work was supported by NIH grant AI-103450 to H Huang. References [1] Tanowitz HB, Machado FS, Jelicks LA, Shirani J, de Carvalho AC, Spray DC, et al. Perspectives on Trypanosoma cruzi-induced heart disease (Chagas disease). Prog Cardiovasc Dis 2009;51:524e39. [2] Zhang L, Tarleton RL. Parasite persistence correlates with disease severity and localization in chronic Chagas' disease. J Infect Dis 1999;180:480e6. [3] Bonney KM, Engman DM. Chagas heart disease pathogenesis: one mechanism or many? Curr Mol Med 2008;8:510e8. [4] Morris SA, Tanowitz HB, Wittner M, Bilezikian JP. Pathophysiological insights into the cardiomyopathy of Chagas' disease. Circulation 1990;82:1900e9. [5] Adesse D, Goldenberg RC, Fortes FS, Jasmin, Iacobas DA, Iacobas S, et al. Gap junctions and Chagas disease. Adv Parasitol 2011;76:63e81. [6] Bhatia V, Garg N. Current status and future prospects for a vaccine against American trypanosomiasis. Expert Rev Vaccines 2005;4:867e80. [7] Bhatia V, Garg N. Previously unrecognized vaccine candidates control Trypanosoma cruzi infection and immunopathology in mice. Clin Vaccine Immunol 2008;15:1158e64.

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