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MOLBIO 10881 1–4
Molecular & Biochemical Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Short communication
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Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi
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Prasad K. Padmanabhan a , Rafael B. Polidoro b , Natasha S. Barteneva c , Ricardo T. Gazzinelli b,d,e , Barbara A. Burleigh a,∗ a
Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, United States Departamento de Bioquímica e Imunologia and Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil c Cellular and Molecular Medicine Program, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, United States d Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brazil e Division of Infectious Disease and Immunology, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, United States b
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Article history: Received 5 January 2015 Received in revised form 6 February 2015 Accepted 14 February 2015 Available online xxx
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Keywords: Trypanosoma cruzi Trypomastigote transfection Fluorescence-activated cell sorting Mammalian cell infection
The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field. © 2015 Published by Elsevier B.V.
The kinetoplastid protozoan parasite, Trypanosoma cruzi, is responsible for human Chagas disease and affects ∼8–10 million people, primarily in the Americas. Chagas disease frequently presents as an aggressive dilated cardiomyopathy for which poor treatment options exist [1]. T. cruzi has a complex life cycle, involving insect vectors for transmission and a wide range of mammalian hosts including an extensive sylvatic reservoir. There are four distinct developmental stages of T. cruzi: epimastigotes and metacyclic trypomastigotes in the insect vector; intracellular amastigotes and bloodstream trypomastigotes in the mammalian host [2]. By virtue of their critical role in tissue infection, persistence and disease progression, the intracellular amastigote forms of T. cruzi represent the most important life cycle stage of the parasite to target for intervention in the host. Despite their clinical relevance, fundamental
∗ Corresponding author at: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Building, Rm 817, 665 Huntington Ave, Boston, MA 02115, United States. Tel.: +1 617 432 2495. E-mail address: [email protected] (B.A. Burleigh).
knowledge of the biology of T. cruzi amastigotes is severely lacking, particularly an understanding of the molecular and cellular processes required to support intracellular replication and survival of these parasites in the host. The genome of T. cruzi was published nearly a decade ago [3]. Since then, genome-scale transcriptomic [4] and proteomic [5] analyses have identified developmentally-regulated genes across T. cruzi life cycle stages that likely reflect functional adaptation to different niches. The ability to translate this type of information into an understanding of critical biochemical and cellular processes in the mammalian-infective stages of T. cruzi has been hampered, in part, by inefficient methods to implement existing molecular genetic tools in this organism. Although far from an exhaustive set of tools, T. cruzi is amenable to molecular genetic manipulations including targeted gene deletion by homologous recombination [6–8] and ectopic expression of transgenes from episomally-maintained plasmids or genomic loci [9–11], with an option for inducible expression [12]. However, the necessity to first perform transfection and selection procedures in the insect vector stage of the parasite – the epimastigotes (Fig. 1A, standard
http://dx.doi.org/10.1016/j.molbiopara.2015.02.001 0166-6851/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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Fig. 1. Transgene expression for rapid protein localization in intracellular T. cruzi amastigotes. (A) An outline of the standard ∼6 weeks protocol for transgene expression and protein localization in intracellular T. cruzi amastigotes (A; standard protocol) contrasted with a streamlined ∼2–3 day protocol that bypasses epimastigote transfection. Direct transfection of trypomastigotes (A; streamlined protocol) with an expression plasmid carrying: (B) GFP alone (pTREXn-GFP) or GFP fused to the C-terminus of (C) T. cruzi histone H1 (pTREXn-histone H1-GFP) or to (D) a putative T. cruzi oxysterol-binding protein (pTREXn-OSBP-GFP) reveals the ability to detect transgene expression in trypomastigotes 18 h post-transfection (B, C) and in intracellular amastigotes (B–D) following infection of HFF with transfected trypomastigotes. Plasmid DNA (10 g) was delivered to purified trypomastigotes (2 × 107 ) in 100 l Tb-BSF buffer (90 mM disodium monophosphate (Na2 HPO4 ), 5 mM potassium chloride, 0.15 mM calcium chloride, 50 mM HEPES, pH 7.3) [19] using an Amaxa nucleofector on setting U-033. Electroporated parasites were diluted in warm DMEM + 10%FBS and placed in the incubator for 30 min to facilitate recovery from electroporation. With the exception of an aliquot of extracellular trypomastigotes that was kept in the 37o C incubator for 18 h (B; extracellular trypomastigotes) the remaining parasites were used to (HFF) in 6-cm2 dishes with glass coverslips. Coverslips were removed and fixed at the times indicated and stained with DAPI to visualize cell and parasite nuclei (blue). Host nuclei are marked with ‘N’. As shown in panel D, OSBP-GFP is clearly visible with distinct subcellular localization by 48 h post-infection (hpi). (E) Estimates of transfection efficiency were obtained by subjecting ∼2 × 106 mock- or pTREXn-GFP-transfected trypomastigotes to flow cytometry using a MACSQuant Analyzer 10 (Miltenyi Biotec® ) at 24 h post-transfection using Flow Jo vX.07 software (TreeStar Inc) for analysis. (F) GFP-positive trypomastigotes were enriched (sorted) from transfected populations (unsorted) 24 h post-transfection by passing parasites resuspended in phenol-free DMEM with 0.5% of BSA through a 15-channel FACSAria II cell sorter (BD Biosciences, San Jose, USA) equipped with a combination of 488 nm, 640 nm, 407 nm and 561 nm lasers was used. Sorted cells were deflected into Falcon tubes containing DMEM-10% FBS. All experimental procedures with non-fixed cells were performed inside a Baker biohood (Baker, Sanford, USA) and according to biosafety BL2+ level practice. To avoid the sorting of cell aggregates, single cells were sequentially selected on FSC-H/FSC-W and SSC-H/SSC-W dot plots. Sorting parameters included: (1) use of an 85 m nozzle; (2) sheath pressure set at 45 psi. To acquire the GFP signal, modified optical filters were used as reported before [20] and the following bandpass (BP) filters were used: 517/20 and/or 514/30 filters from Semrock Inc (Rochester, NY, USA). As a dichroic filter a 502 longpass filter (502LP) from same company was used. To exclude auto-fluorescing cells and improve signal-to-noise ratio additional gating on the violet channel (optical filter 450/50; excitation laser 407 nm) was applied. Infection of HFF monolayers with trypomastigotes enriched for GFP expression by cell sorting (sorted) yielded populations of intracellular amastigotes that were >90% GFP-positive, as compared to ∼5% GFP-positive amastigotes arising from infections with (unsorted) transfected trypomastigotes. The GFP was fused with respective genes by Phusion® High-Fidelity DNA Polymerase (NEB) using specific primers for fusion PCR amplification and cloning of GFP chimeras into the pTREXn vector. For pTREXn-histone H1-GFP: Forward: 5 -GCTCTAGAATGCTAAACGCGGACCCGTTG-3 ; Reverse: 5 -GTGAAAAGTTCTTCTCCTTTACTCTTCTTCGGCGCCTTCTTCA-3 ; GFP: Forward: 5 -AGTAAAGGAGAAGAACTTTTCAC-3 ; Reverse: 5 -CCGCTCGAGTTATTTGTATAGTTCATCCATGC-3 . pTREXn-OSBP-GFP: Forward: 5 -GCTCTAGAATGGCGAAAACCAGTCG-3 ; Reverse: 5 -GTGAAAAGTTCTTCTCCTTTACTATTCAGCAACATATCCCGTG-3 and GFP was amplified using specific primers as above.
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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protocol) [13,14] – adds weeks of time to protocols that are ultimately aimed at studying the biology of the mammalian-infective stages of T. cruzi. Driven by the need to access intracellular T. cruzi amastigote stages more readily, we tested the ability to deliver plasmid DNA to the non-dividing tissue culture trypomastigote forms of the parasite (Fig. 1A, streamlined protocol), such that upon mammalian cell infection, transgenes are expressed and localized in the intracellular amastigotes. The feasibility of this approach is demonstrated by exploiting the T. cruzi expression vector pTREXn [10], engineered to express green fluorescent protein (GFP) alone (Fig. 1B) or as C-terminal fusions with T. cruzi histone H1 (TriTrypDB.org; TcCLB.510225.10) (Fig. 1C) or with the putative T. cruzi oxysterolbinding protein (TriTrypDB.org; TcCLB.508211.10) (Fig. 1D). Tissue culture trypomastigotes, isolated from culture supernatants of infected LLcMK2 cells, as described [15], were washed, adjusted to ∼2 × 107 parasites in 100 l electroporation buffer and pulsed with 10 g plasmid DNA using an Amaxa® Nucleofector (see legend to Fig. 1 for detail). Immediately following transfection, trypomastigotes were diluted in 8 ml of warm DMEM medium containing 10% FBS (DMEM-10) and incubated at 37 ◦ C in 5% CO2 for 30 min to facilitate recovery. NB: performing this step with serum concentrations lower than 10% resulted in reduced recovery and lower infectivity (data not shown). Trypomastigotes were then distributed evenly into 8 wells of a 24-well plate containing monolayers of human foreskin fibroblasts (HFF) grown on 12 mm round glass coverslips. Parasites were incubated with cells for 18 h to promote maximal host cell invasion and conversion to intracellular amastigotes. Remaining extracellular parasites were removed by extensive washing in PBS and fresh DMEM-10 was added for the duration of the incubation period. In order to follow transgene expression in the trypomastigote stage itself, aliquots containing ∼2 × 106 transfected trypomastigotes were routinely retained (i.e. not used for mammalian cell infection) and incubated axenically for 18–24 h in DMEM-10 at 37 ◦ C, 5% CO2 . Following transfection with pTREXn-GFP, cytosolic GFP expression in trypomastigotes was evident at 18 h posttransfection by epifluorescence microscopy (e.g. Fig. 1B and C) and in intracellular T. cruzi amastigotes (Fig. 1B–D), as early as 24 hpi (not shown). GFP expression is readily observed in clusters of replicating intracellular amastigotes (Fig. 1B; intracellular amastigote 72 hpi) and in intracellular trypomastigotes that develop toward the end of the mammalian cell infection cycle (Fig. 1B; intracellular trypomastigote; 120 hpi). Approximately 50–100 infected host cells containing GFP-expressing parasites were observed per coverslip. Therefore, from a single transfection, starting with 2 × 107 trypomastigotes we were able to observe transgene-expressing intracellular amastigotes at several different time points. A major motivation for improving existing protocols for ectopic protein expression in intracellular T. cruzi amastigotes is to expedite subcellular localization and characterization of novel parasite proteins. Here, we demonstrate the ability to perform rapid localization of GFP-fusion proteins in the mammalian-infective stages of T. cruzi. Following transfection of T. cruzi trypomastigotes with pTREXn-Histone1-GFP, the predicted nuclear localization of GFP is observed [16] in both axenic trypomastigotes and intracellular amastigotes (Fig. 1C). Next, we examined a previously uncharacterized T. cruzi protein that has a predicted N-terminal transmembrane domain and a putative oxysterol-binding protein domain (OSBP; Fig. 1D). OSBP-GFP localizes to subcellular, punctate structures outside of the parasite nucleus and kinetoplast (Fig. 1D) but becomes cytosolic in the absence of the predicted transmembrane domain (not shown). Similar localization of OSBP was obtained when GFP was replaced with the epitope tags, HA or Ty-1, followed by immunofluorescence microscopy, and in stable T. cruzi transfectants derived from transfected epimastigotes (not shown). These
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examples provide a clear demonstration that direct trypomastigote transfection is a viable method for the transient expression and rapid localization of proteins of interest in T. cruzi amastigotes – in the context of a mammalian host cell infection. While the localization of any ectopically-expressed protein always requires independent validation, our method opens the door to rapid screening and initial characterization of differentially-expressed and hypothetical proteins in the clinically relevant stages of T. cruzi. To estimate transfection efficiency, axenic trypomastigotes maintained in DMEM-10 at 37 ◦ C in 5% CO2 overnight following transfection were subjected to flow cytometry (Fig. 1E) and to manual counting using the epifluorescence microscope 24 h after delivery of the pTREXn-GFP plasmid. Log-phase epimastigotes, maintained as described [6], were transfected in parallel for comparison. Mock-infected controls of both life cycle stages were included to gate for auto-fluorescence (Fig. 1E; left panel). In repeated experiments, we observe that ∼5% of the viable trypomastigotes recovered at 24 h post-transfection express GFP (Fig. 1E, right panel). The transfection efficiency of viable epimastigotes was ∼16% (not shown). These values were confirmed independently by fluorescence microscopy (not shown). It should be noted that estimates of trypomastigote transfection efficiency are likely to be underestimated as overnight incubation of trypomastigotes in liquid culture results in some loss of viability and spontaneous conversion to amastigotes. These parasites would be gated out in analysis of the flow cytometry data. Our observation that the relative number of GFP-expressing amastigotes present in mammalian cells at 18 hpi (a pre-replication time point) is consistently higher than the estimated number of transfected trypomastigotes representative of the infecting population, supports this assertion. This also suggests that there is no overt selection against DNAtransfected trypomastigotes during the host cell invasion process and during early amastigote development in mammalian cells. The ability to recover viable GFP-expressing T. cruzi trypomastigotes encouraged us to pursue the enrichment of this population by fluorescence-activated cell sorting (FACS), which has been successfully applied to other kinetoplastid protozoan parasites [17,18]. Trypomastigotes, incubated axenically for 24 h following transfection, were passed through a 15-channel FACSAria II cell sorter and GFP-positive parasites collected. After sorting, ∼98% of the recovered parasites were GFP-positive by fluorescence microscopy and 90–95% of the population was viable as determined by motility. Sorted GFP-positive parasites were used to infect HFF monolayers in parallel with transfected but unsorted parasites from the same experiment. As anticipated, only ∼5% of the intracellular amastigotes arising in cell monolayers at 48 hpi from unsorted parasites were visibly GFP-positive (Fig. 1F, left panel). In contrast, 90% of the intracellular amastigotes resulting from sorted trypomastigote populations expressed GFP (Fig. 1F, right panel). As infection progresses, the amastigotes eventually give rise to GFP-expressing trypomastigotes that are released from cells and can be used to infect fresh host cell monolayers. However, in the absence of drug selection to maintain the pTREXn-GFP plasmid the parasites lose GFP expression rapidly. We are currently exploring methods to select for stable T. cruzi transfectants in mammalian cell monolayers, which would open the door to a complete by-pass of epimastigotes for many genetic manipulations in T. cruzi in which the desired outcome is to study mammalian-infective stages of the parasite. In summary, the ability to achieve transfection efficiencies of ∼5% in T. cruzi trypomastigotes, with excellent post-transfection viability and infectivity, allows rapid and direct evaluation of transgene expression with subcellular localization in the mammalian-infective stages of T. cruzi. The ability to achieve this within 2–3 days, rather than in weeks using standard protocols, presents unprecedented opportunities for rapid ‘screening’ of novel
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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proteins in intracellular T. cruzi amastigotes. Given that parasite stage-specific expression data is a growing resource in the Chagas disease field, the addition of a practical tool for rapid preliminary analysis of developmentally-regulated genes in T. cruzi is timely. This approach may be particularly valuable for the large class of hypothetical proteins that comprise the predicted T. cruzi proteome. The inclusion of an optional FACS-based enrichment step for fluorescent trypomastigotes, prior to host cell infection, increases the number of transgene-expressing intracellular T. cruzi amastigotes to >90%. In addition to generating a significantly larger amastigote population for microscopic analysis (Fig. 1F), initiation of mammalian cell infections with sorted parasites creates potential opportunities to conduct comparative biochemical and/or transcriptomic analyses, where both parasite and host can be probed for the impact of parasite transgene expression. In addition, FACS-based enrichment of T. cruzi transfectants offers the potential to exploit drug resistance cassettes carried on plasmids to select for stably transfected T. cruzi amastigotes directly in mammalian host cell monolayers. With several potential applications, this expedited protocol for transfection and transgene expression in the mammalian-infective forms of T. cruzi is predicted to facilitate biological discovery in the Chagas disease field. 1. Conflict of interest The authors declare no conflict of interest. Acknowledgements
We thank members of the Burleigh lab and the Molecular Para220 sitology group at HSPH for helpful discussions. R.P.B. was supported 221 Q4 by CNPq fellowship #219331/2013-8. R.T.G. was a recipient of 222 a Scholar Fellowship from Coordenac¸ão de Aperfeic¸oamento de 223 Pessoal de Ensino Superior (CAPES), Brazil, and the David Rocke224 feller Center for Latin American Studies at Harvard School of Public 225 Health, USA. 219
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References [1] Coura JR, Vinas PA. Chagas disease: a new worldwide challenge. Nature 2010;465:S6–7, http://dx.doi.org/10.1038/nature09221. [2] Brener Z. Life cycle of Trypanosoma cruzi. Rev Inst Med Trop Sao Paulo 1971;13:171–8.
[3] El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, et al. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 2005;309:409–15. [4] Minning TA, Weatherly DB, Atwood III J, Orlando R, Tarleton RL. The steadystate transcriptome of the four major life-cycle stages of Trypanosoma cruzi. BMC Genomics 2009;10:370, http://dx.doi.org/10.1186/1471-2164-10-370. [5] Atwood III JA, Weatherly DB, Minning TA, Bundy B, Cavola C, Opperdoes FR, et al. The Trypanosoma cruzi proteome. Science 2005;309:473–6. [6] Caler EV, Vaena de Avalos S, Haynes PA, Andrews NW, Burleigh BA. Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J 1998;17:4975–86. [7] Garzon E, Borges MC, Cordeiro-da-Silva A, Nacife V, Meirelles Mde N, Guilvard E, et al. Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele elicits attenuated Chagas’ disease in mice. Immunol Lett 2003;89:67–80. [8] Perez Brandan C, Basombrio MA. Genetically attenuated Trypanosoma cruzi parasites as a potential vaccination tool. Bioengineered 2012;3:242–6, http://dx.doi.org/10.4161/bioe.20680. [9] Vazquez MP, Levin MJ. Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene 1999;239:217–25. [10] Lorenzi HA, Vazquez MP, Levin MJ. Integration of expression vectors into the ribosomal locus of Trypanosoma cruzi. Gene 2003;310:91–9. [11] DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, et al. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 2004;92:113–20. [12] Taylor MC, Kelly JM. pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi. BMC Biotechnol 2006;6:32. [13] Kelly JM, Ward HM, Miles MA, Kendall G. A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res 1992;20:3963–9. [14] Lu HY, Buck GA. Expression of an exogenous gene in Trypanosoma cruzi epimastigotes. Mol Biochem Parasitol 1991;44:109–14. [15] Caradonna KL, Engel JC, Jacobi D, Lee CH, Burleigh BA. Host metabolism regulates intracellular growth of Trypanosoma cruzi. Cell Host Microbe 2013;13:108–17, http://dx.doi.org/10.1016/j.chom.2012.11.011. [16] Gutiyama LM, da Cunha JP, Schenkman S. Histone H1 of Trypanosoma cruzi is concentrated in the nucleolus region and disperses upon phosphorylation during progression to mitosis. Eukaryot Cell 2008;7:560–8, http://dx.doi.org/10.1128/EC.00460-07. [17] Ha DS, Schwarz JK, Turco SJ, Beverley SM. Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 1996;77:57–64. [18] Bauer S, Morris JC, Morris MT. Environmentally regulated glycosome protein composition in the African trypanosome. Eukaryot Cell 2013;12:1072–9, http://dx.doi.org/10.1128/EC.00086-13. [19] Burkard G, Fragoso CM, Roditi I. Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. Mol Biochem Parasitol 2007;153: 220–3. [20] Vorobjev IA, Buchholz K, Prabhat P, Ketman K, Egan ES, Marti M, et al. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters. Malar J 2012;11:312, http://dx.doi.org/10.1186/1475-2875-11-312.
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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ARTICLE IN PRESS
MOLBIO 10881 1–4
Molecular & Biochemical Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Short communication
1
Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi
2
3
4
Q1
5 6
Q2
7 8 9 10 11 12
Prasad K. Padmanabhan a , Rafael B. Polidoro b , Natasha S. Barteneva c , Ricardo T. Gazzinelli b,d,e , Barbara A. Burleigh a,∗ a
Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, United States Departamento de Bioquímica e Imunologia and Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil c Cellular and Molecular Medicine Program, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, United States d Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brazil e Division of Infectious Disease and Immunology, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, United States b
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Article history: Received 5 January 2015 Received in revised form 6 February 2015 Accepted 14 February 2015 Available online xxx
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Keywords: Trypanosoma cruzi Trypomastigote transfection Fluorescence-activated cell sorting Mammalian cell infection
The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field. © 2015 Published by Elsevier B.V.
The kinetoplastid protozoan parasite, Trypanosoma cruzi, is responsible for human Chagas disease and affects ∼8–10 million people, primarily in the Americas. Chagas disease frequently presents as an aggressive dilated cardiomyopathy for which poor treatment options exist [1]. T. cruzi has a complex life cycle, involving insect vectors for transmission and a wide range of mammalian hosts including an extensive sylvatic reservoir. There are four distinct developmental stages of T. cruzi: epimastigotes and metacyclic trypomastigotes in the insect vector; intracellular amastigotes and bloodstream trypomastigotes in the mammalian host [2]. By virtue of their critical role in tissue infection, persistence and disease progression, the intracellular amastigote forms of T. cruzi represent the most important life cycle stage of the parasite to target for intervention in the host. Despite their clinical relevance, fundamental
∗ Corresponding author at: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Building, Rm 817, 665 Huntington Ave, Boston, MA 02115, United States. Tel.: +1 617 432 2495. E-mail address: [email protected] (B.A. Burleigh).
knowledge of the biology of T. cruzi amastigotes is severely lacking, particularly an understanding of the molecular and cellular processes required to support intracellular replication and survival of these parasites in the host. The genome of T. cruzi was published nearly a decade ago [3]. Since then, genome-scale transcriptomic [4] and proteomic [5] analyses have identified developmentally-regulated genes across T. cruzi life cycle stages that likely reflect functional adaptation to different niches. The ability to translate this type of information into an understanding of critical biochemical and cellular processes in the mammalian-infective stages of T. cruzi has been hampered, in part, by inefficient methods to implement existing molecular genetic tools in this organism. Although far from an exhaustive set of tools, T. cruzi is amenable to molecular genetic manipulations including targeted gene deletion by homologous recombination [6–8] and ectopic expression of transgenes from episomally-maintained plasmids or genomic loci [9–11], with an option for inducible expression [12]. However, the necessity to first perform transfection and selection procedures in the insect vector stage of the parasite – the epimastigotes (Fig. 1A, standard
http://dx.doi.org/10.1016/j.molbiopara.2015.02.001 0166-6851/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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Fig. 1. Transgene expression for rapid protein localization in intracellular T. cruzi amastigotes. (A) An outline of the standard ∼6 weeks protocol for transgene expression and protein localization in intracellular T. cruzi amastigotes (A; standard protocol) contrasted with a streamlined ∼2–3 day protocol that bypasses epimastigote transfection. Direct transfection of trypomastigotes (A; streamlined protocol) with an expression plasmid carrying: (B) GFP alone (pTREXn-GFP) or GFP fused to the C-terminus of (C) T. cruzi histone H1 (pTREXn-histone H1-GFP) or to (D) a putative T. cruzi oxysterol-binding protein (pTREXn-OSBP-GFP) reveals the ability to detect transgene expression in trypomastigotes 18 h post-transfection (B, C) and in intracellular amastigotes (B–D) following infection of HFF with transfected trypomastigotes. Plasmid DNA (10 g) was delivered to purified trypomastigotes (2 × 107 ) in 100 l Tb-BSF buffer (90 mM disodium monophosphate (Na2 HPO4 ), 5 mM potassium chloride, 0.15 mM calcium chloride, 50 mM HEPES, pH 7.3) [19] using an Amaxa nucleofector on setting U-033. Electroporated parasites were diluted in warm DMEM + 10%FBS and placed in the incubator for 30 min to facilitate recovery from electroporation. With the exception of an aliquot of extracellular trypomastigotes that was kept in the 37o C incubator for 18 h (B; extracellular trypomastigotes) the remaining parasites were used to (HFF) in 6-cm2 dishes with glass coverslips. Coverslips were removed and fixed at the times indicated and stained with DAPI to visualize cell and parasite nuclei (blue). Host nuclei are marked with ‘N’. As shown in panel D, OSBP-GFP is clearly visible with distinct subcellular localization by 48 h post-infection (hpi). (E) Estimates of transfection efficiency were obtained by subjecting ∼2 × 106 mock- or pTREXn-GFP-transfected trypomastigotes to flow cytometry using a MACSQuant Analyzer 10 (Miltenyi Biotec® ) at 24 h post-transfection using Flow Jo vX.07 software (TreeStar Inc) for analysis. (F) GFP-positive trypomastigotes were enriched (sorted) from transfected populations (unsorted) 24 h post-transfection by passing parasites resuspended in phenol-free DMEM with 0.5% of BSA through a 15-channel FACSAria II cell sorter (BD Biosciences, San Jose, USA) equipped with a combination of 488 nm, 640 nm, 407 nm and 561 nm lasers was used. Sorted cells were deflected into Falcon tubes containing DMEM-10% FBS. All experimental procedures with non-fixed cells were performed inside a Baker biohood (Baker, Sanford, USA) and according to biosafety BL2+ level practice. To avoid the sorting of cell aggregates, single cells were sequentially selected on FSC-H/FSC-W and SSC-H/SSC-W dot plots. Sorting parameters included: (1) use of an 85 m nozzle; (2) sheath pressure set at 45 psi. To acquire the GFP signal, modified optical filters were used as reported before [20] and the following bandpass (BP) filters were used: 517/20 and/or 514/30 filters from Semrock Inc (Rochester, NY, USA). As a dichroic filter a 502 longpass filter (502LP) from same company was used. To exclude auto-fluorescing cells and improve signal-to-noise ratio additional gating on the violet channel (optical filter 450/50; excitation laser 407 nm) was applied. Infection of HFF monolayers with trypomastigotes enriched for GFP expression by cell sorting (sorted) yielded populations of intracellular amastigotes that were >90% GFP-positive, as compared to ∼5% GFP-positive amastigotes arising from infections with (unsorted) transfected trypomastigotes. The GFP was fused with respective genes by Phusion® High-Fidelity DNA Polymerase (NEB) using specific primers for fusion PCR amplification and cloning of GFP chimeras into the pTREXn vector. For pTREXn-histone H1-GFP: Forward: 5 -GCTCTAGAATGCTAAACGCGGACCCGTTG-3 ; Reverse: 5 -GTGAAAAGTTCTTCTCCTTTACTCTTCTTCGGCGCCTTCTTCA-3 ; GFP: Forward: 5 -AGTAAAGGAGAAGAACTTTTCAC-3 ; Reverse: 5 -CCGCTCGAGTTATTTGTATAGTTCATCCATGC-3 . pTREXn-OSBP-GFP: Forward: 5 -GCTCTAGAATGGCGAAAACCAGTCG-3 ; Reverse: 5 -GTGAAAAGTTCTTCTCCTTTACTATTCAGCAACATATCCCGTG-3 and GFP was amplified using specific primers as above.
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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protocol) [13,14] – adds weeks of time to protocols that are ultimately aimed at studying the biology of the mammalian-infective stages of T. cruzi. Driven by the need to access intracellular T. cruzi amastigote stages more readily, we tested the ability to deliver plasmid DNA to the non-dividing tissue culture trypomastigote forms of the parasite (Fig. 1A, streamlined protocol), such that upon mammalian cell infection, transgenes are expressed and localized in the intracellular amastigotes. The feasibility of this approach is demonstrated by exploiting the T. cruzi expression vector pTREXn [10], engineered to express green fluorescent protein (GFP) alone (Fig. 1B) or as C-terminal fusions with T. cruzi histone H1 (TriTrypDB.org; TcCLB.510225.10) (Fig. 1C) or with the putative T. cruzi oxysterolbinding protein (TriTrypDB.org; TcCLB.508211.10) (Fig. 1D). Tissue culture trypomastigotes, isolated from culture supernatants of infected LLcMK2 cells, as described [15], were washed, adjusted to ∼2 × 107 parasites in 100 l electroporation buffer and pulsed with 10 g plasmid DNA using an Amaxa® Nucleofector (see legend to Fig. 1 for detail). Immediately following transfection, trypomastigotes were diluted in 8 ml of warm DMEM medium containing 10% FBS (DMEM-10) and incubated at 37 ◦ C in 5% CO2 for 30 min to facilitate recovery. NB: performing this step with serum concentrations lower than 10% resulted in reduced recovery and lower infectivity (data not shown). Trypomastigotes were then distributed evenly into 8 wells of a 24-well plate containing monolayers of human foreskin fibroblasts (HFF) grown on 12 mm round glass coverslips. Parasites were incubated with cells for 18 h to promote maximal host cell invasion and conversion to intracellular amastigotes. Remaining extracellular parasites were removed by extensive washing in PBS and fresh DMEM-10 was added for the duration of the incubation period. In order to follow transgene expression in the trypomastigote stage itself, aliquots containing ∼2 × 106 transfected trypomastigotes were routinely retained (i.e. not used for mammalian cell infection) and incubated axenically for 18–24 h in DMEM-10 at 37 ◦ C, 5% CO2 . Following transfection with pTREXn-GFP, cytosolic GFP expression in trypomastigotes was evident at 18 h posttransfection by epifluorescence microscopy (e.g. Fig. 1B and C) and in intracellular T. cruzi amastigotes (Fig. 1B–D), as early as 24 hpi (not shown). GFP expression is readily observed in clusters of replicating intracellular amastigotes (Fig. 1B; intracellular amastigote 72 hpi) and in intracellular trypomastigotes that develop toward the end of the mammalian cell infection cycle (Fig. 1B; intracellular trypomastigote; 120 hpi). Approximately 50–100 infected host cells containing GFP-expressing parasites were observed per coverslip. Therefore, from a single transfection, starting with 2 × 107 trypomastigotes we were able to observe transgene-expressing intracellular amastigotes at several different time points. A major motivation for improving existing protocols for ectopic protein expression in intracellular T. cruzi amastigotes is to expedite subcellular localization and characterization of novel parasite proteins. Here, we demonstrate the ability to perform rapid localization of GFP-fusion proteins in the mammalian-infective stages of T. cruzi. Following transfection of T. cruzi trypomastigotes with pTREXn-Histone1-GFP, the predicted nuclear localization of GFP is observed [16] in both axenic trypomastigotes and intracellular amastigotes (Fig. 1C). Next, we examined a previously uncharacterized T. cruzi protein that has a predicted N-terminal transmembrane domain and a putative oxysterol-binding protein domain (OSBP; Fig. 1D). OSBP-GFP localizes to subcellular, punctate structures outside of the parasite nucleus and kinetoplast (Fig. 1D) but becomes cytosolic in the absence of the predicted transmembrane domain (not shown). Similar localization of OSBP was obtained when GFP was replaced with the epitope tags, HA or Ty-1, followed by immunofluorescence microscopy, and in stable T. cruzi transfectants derived from transfected epimastigotes (not shown). These
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examples provide a clear demonstration that direct trypomastigote transfection is a viable method for the transient expression and rapid localization of proteins of interest in T. cruzi amastigotes – in the context of a mammalian host cell infection. While the localization of any ectopically-expressed protein always requires independent validation, our method opens the door to rapid screening and initial characterization of differentially-expressed and hypothetical proteins in the clinically relevant stages of T. cruzi. To estimate transfection efficiency, axenic trypomastigotes maintained in DMEM-10 at 37 ◦ C in 5% CO2 overnight following transfection were subjected to flow cytometry (Fig. 1E) and to manual counting using the epifluorescence microscope 24 h after delivery of the pTREXn-GFP plasmid. Log-phase epimastigotes, maintained as described [6], were transfected in parallel for comparison. Mock-infected controls of both life cycle stages were included to gate for auto-fluorescence (Fig. 1E; left panel). In repeated experiments, we observe that ∼5% of the viable trypomastigotes recovered at 24 h post-transfection express GFP (Fig. 1E, right panel). The transfection efficiency of viable epimastigotes was ∼16% (not shown). These values were confirmed independently by fluorescence microscopy (not shown). It should be noted that estimates of trypomastigote transfection efficiency are likely to be underestimated as overnight incubation of trypomastigotes in liquid culture results in some loss of viability and spontaneous conversion to amastigotes. These parasites would be gated out in analysis of the flow cytometry data. Our observation that the relative number of GFP-expressing amastigotes present in mammalian cells at 18 hpi (a pre-replication time point) is consistently higher than the estimated number of transfected trypomastigotes representative of the infecting population, supports this assertion. This also suggests that there is no overt selection against DNAtransfected trypomastigotes during the host cell invasion process and during early amastigote development in mammalian cells. The ability to recover viable GFP-expressing T. cruzi trypomastigotes encouraged us to pursue the enrichment of this population by fluorescence-activated cell sorting (FACS), which has been successfully applied to other kinetoplastid protozoan parasites [17,18]. Trypomastigotes, incubated axenically for 24 h following transfection, were passed through a 15-channel FACSAria II cell sorter and GFP-positive parasites collected. After sorting, ∼98% of the recovered parasites were GFP-positive by fluorescence microscopy and 90–95% of the population was viable as determined by motility. Sorted GFP-positive parasites were used to infect HFF monolayers in parallel with transfected but unsorted parasites from the same experiment. As anticipated, only ∼5% of the intracellular amastigotes arising in cell monolayers at 48 hpi from unsorted parasites were visibly GFP-positive (Fig. 1F, left panel). In contrast, 90% of the intracellular amastigotes resulting from sorted trypomastigote populations expressed GFP (Fig. 1F, right panel). As infection progresses, the amastigotes eventually give rise to GFP-expressing trypomastigotes that are released from cells and can be used to infect fresh host cell monolayers. However, in the absence of drug selection to maintain the pTREXn-GFP plasmid the parasites lose GFP expression rapidly. We are currently exploring methods to select for stable T. cruzi transfectants in mammalian cell monolayers, which would open the door to a complete by-pass of epimastigotes for many genetic manipulations in T. cruzi in which the desired outcome is to study mammalian-infective stages of the parasite. In summary, the ability to achieve transfection efficiencies of ∼5% in T. cruzi trypomastigotes, with excellent post-transfection viability and infectivity, allows rapid and direct evaluation of transgene expression with subcellular localization in the mammalian-infective stages of T. cruzi. The ability to achieve this within 2–3 days, rather than in weeks using standard protocols, presents unprecedented opportunities for rapid ‘screening’ of novel
Please cite this article in press as: Padmanabhan PK, et al. Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol (2015), http://dx.doi.org/10.1016/j.molbiopara.2015.02.001
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proteins in intracellular T. cruzi amastigotes. Given that parasite stage-specific expression data is a growing resource in the Chagas disease field, the addition of a practical tool for rapid preliminary analysis of developmentally-regulated genes in T. cruzi is timely. This approach may be particularly valuable for the large class of hypothetical proteins that comprise the predicted T. cruzi proteome. The inclusion of an optional FACS-based enrichment step for fluorescent trypomastigotes, prior to host cell infection, increases the number of transgene-expressing intracellular T. cruzi amastigotes to >90%. In addition to generating a significantly larger amastigote population for microscopic analysis (Fig. 1F), initiation of mammalian cell infections with sorted parasites creates potential opportunities to conduct comparative biochemical and/or transcriptomic analyses, where both parasite and host can be probed for the impact of parasite transgene expression. In addition, FACS-based enrichment of T. cruzi transfectants offers the potential to exploit drug resistance cassettes carried on plasmids to select for stably transfected T. cruzi amastigotes directly in mammalian host cell monolayers. With several potential applications, this expedited protocol for transfection and transgene expression in the mammalian-infective forms of T. cruzi is predicted to facilitate biological discovery in the Chagas disease field. 1. Conflict of interest The authors declare no conflict of interest. Acknowledgements
We thank members of the Burleigh lab and the Molecular Para220 sitology group at HSPH for helpful discussions. R.P.B. was supported 221 Q4 by CNPq fellowship #219331/2013-8. R.T.G. was a recipient of 222 a Scholar Fellowship from Coordenac¸ão de Aperfeic¸oamento de 223 Pessoal de Ensino Superior (CAPES), Brazil, and the David Rocke224 feller Center for Latin American Studies at Harvard School of Public 225 Health, USA. 219
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