Simplified inducible system for Trypanosoma brucei

Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem “head-to-tail” configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including α-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.


Introduction
Genetic manipulation tools for Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, have appropriately been tailored to suit the peculiar regulation of gene expression found in kinetoplastids. In the genome, unrelated protein coding genes are arranged in long units and regulation takes place mainly at the post-transcriptional level [1,2]. Transcription by RNA polymerase II (RNAPII) produces polycistronic RNA molecules [3,4] and transcribed intergenic sequences participate in the trans-splicing between the downstream gene and the cap bearing spliced leader, which is transcribed elsewhere in the genome. In addition this processing is also responsible in a concerted manner for the polyadenylation of the upstream coding sequence, thus resulting in the maturation of each mRNA [5,6]. Furthermore, the remnants of the transcribed intergenic sequence, namely the 5´and 3´untranslated regions determine messenger stability and steady state levels as well as translation efficiency through the binding of other cellular components [7][8][9][10]. Hence in genetic constructs PLOS  selection steps as well as lengthy periods in culture can affect the natural wild type properties sought after in field isolates. On the other hand, given the limited number of antibiotic resistance genes available for trypanosome selection, inducible systems that minimize the required number of these elements are highly desirable. To some extent, this issue has been dealt with in works that extend the possibilities for further genetic manipulation methodologies [19,20,23]. Moreover, the recent development of an episome-based CRISPR-Cas9 system has allowed genome edition without the insertion of a resistance marker [34], underscoring the interest in methodologies that reduce or circumvent the use of these elements.
In this work we have developed an alternative simplified inducible system based entirely on a single plasmid. It has been designed to contain the sequence of interest along with all other required regulatory elements avoiding the need for pre-established cell lines.

Plasmid construction
For the construction of pTbFIX and pTbFIX-PARP several fragments were PCR-amplified, cloned into pGEM-T Easy (Promega Corporation, Madison, WI, USA) and sequenced (Macrogen, Seoul, Korea). The tetR gene was derived from pLEW13 [17] and the sequence for the c-terminal NLS was added in frame to the 3´primer. The pLEW100v5 vector (Dr. George Cross Lab, [17]) obtained from Addgene (Cambridge, MA, USA) was used as template for the amplification of the constitutive and inducible versions of the rRNA promoter, as well as the ribosomal spacer targeting sequence and aldolase 3´intergenic region. Other fragments (pUC min backbone vector, PARP promoter, mEGFP gene and actin and α/β-tubulin intergenic sequences) were derived from already available plasmids. A detailed description of the performed cloning steps can be obtained upon request. The sequences of both vectors were deposited in GenBank under accession numbers MH936667 and MH936668.
Constructs for RNAi gene silencing: For the construction of pTbFIX-αT, Lister 427 genomic DNA was used as template for the PCR-amplification of 550 bp and 864 bp segments from the α-tubulin genes. In each case the same forward (AAGCTTGCTAGCCACTACACCATTGG TAAGGAG; HindIII site underlined and NheI site in bold) primer and two alternative reverse primers (GGGCCCGAGACAGAGAGTTGCTCGTGG and GGGCCCTGTGGTCAATACGAGCGAAC; PspOMI site underlined) were used respectively. After cloning into pGEM-T Easy and being sequenced, the HindIII/PspOMI shorter and the NheI/PspOMI longer segments were inserted into HindIII/SpeI digested pTbFIX producing pTbFIX-αT by means of a three fragment ligation. A 1553 bp HindIII/PstI segment from the latter was transferred to pTbFIX-PARP thus giving rise to pTbFIX-PARP-αT. An equivalent strategy was employed for the generation of constructs allowing the RNAi silencing of enolase and clathrin heavy chain genes (pTbFIX-ENO and pTbFIX-CLH). With the same forward primer (AAGCTTGGATCCACGATCCAGAA AGTTCACGGTC; HindIII site underlined and BamHI site in bold) and two reverse primers (TCTAGACCTCAGAACCCATACGCAG and TCTAGAAGCGGCTCATTAATGTCTTTG; XbaI site underlined) 553 bp and 662 bp long fragments from the enolase gene were amplified. Likewise with the forward (AAGCTTGGATCCGATTAACCTGAAGCATTCCCAT; HindIII site underlined and BamHI site in bold) and both reverse (TCTAGAACACGTTAATTAGTTT GTCCGCA and TCTAGAGTTCCTGCACCTGCCCAAC; XbaI site underlined) primers 377 bp and 503 bp segments from the CLH gene were cloned. The three fragment ligations into Hin-dIII/BamHI digested pTbFIX involved the shorter HindIII/XbaI fragment with the corresponding BamHI/XbaI longer fragment. All primers used in this work were ordered from Macrogen.

Generation of transgenic trypanosome cell lines
For each transfection, approximately 2×10 7 BSF trypanosomes grown to mid log phase (~0.8-1.0x10 6 cells/mL) in HMI-9 medium were collected, washed once with cytomix [36] (2 mM EGTA, 120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 pH 7.6, 25 mM HEPES pH 7.6, 5 mM MgCl 2 .6H 2 O) modified with 0.5% glucose, 100 μg/mL BSA and 1 mM hypoxanthine at room temperature, and resuspended in 500 μl of the same buffer. About 5-15 μg of linearized plasmid was mixed with the cell suspension and transferred to a BTX 0.2 cm electroporation cuvette. Electroporation was immediately performed at a single pulse with a BTX 600 Electro Cell Manipulator (BTX Inc., San Diego, CA, USA) at 1.1 kV, 25 μF and R6 resistance (186 Ω). The entire cell-DNA mixture was transferred to a flask containing 5 mL of fresh HMI-9 medium and incubated for 6 hours at 37˚C without selection. Cells were diluted to a density of 2x10 5 cell/mL and 1 mL cell aliquots were distributed in 24-well plates. Selection was achieved by the addition of 0.1 μg/mL of puromycin (InvivoGen).
PCF cells (approximately 2×10 7 trypanosomes) were washed and resuspended in 500 μl of modified cytomix medium at 4˚C. Parasites were electroporated using a single pulse at a voltage of 1.4 kV, 25 μF capacitance and 24 Ω resistance. Selection was achieved by the addition of 1 μg/mL of puromycin.
Blots were probed with Alexa Fluor 790 AffiniPure Goat Anti-Mouse IgG (H+L), Alexa Fluor 680 AffiniPure Goat Anti-Mouse IgG (H+L) or Alexa Fluor 680 AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Signal intensities were detected using an Odyssey laser-scanning system and quantified with Image Studio software (LI-COR Biosciences, Lincoln, NE, USA). Prestained Protein Molecular Weight markers used were from Pierce (Rockford, IL, USA).

Plasmid description
The system conceived for fast inducible expression in T. brucei is based on vector pTbFIX ( Fig  1). The plasmid contains two transcription units. The first drives the expression of a green fluorescent protein (GFP) reporter gene through an inducible rRNA promoter. Downstream, a constitutive dicistronic unit encoding a nucleus targeted tetR and puromycin resistance genes was inserted. To study the effect of different tetR abundances on GFP expression regulation, the original constitutive rRNA promoter in pTbFIX was replaced by that of PARP, thus giving rise to pTbFIX-PARP. After linearization at a unique NotI restriction site inside a targeting sequence, the construct is directed to the non-transcribed spacer of ribosomal RNA gene repeats for integration by single crossover "ends-in" homologous recombination.

Inducible GFP expression in PCF
Both pTbFIX and pTbFIX-PARP vectors where transfected into wild-type Lister 427 procyclic form trypomastigotes and transgenic cell lines were easily obtained. After selection, the resulting parasite populations were cultured with 1 μg/mL of doxycycline which corresponds to the highest concentration employed for other tetR based inducible systems in order to obtain maximal induction [17,21]. Doubling times of induced and uninduced cells were similar to those of the wild-type parental strain indicating that the constructs had no effect on cellular viability (S1 Fig). Microscopic examination showed a homogeneous population of fluorescent cells in samples derived from induced cultures (Fig 2A). In contrast, no GFP signal could be detected in uninduced equivalents. Protein samples were analyzed by Western blot with antibodies raised against tetR. In uninduced conditions the constitutive rRNA promoter produced higher (4.2-fold) repressor amounts than those obtained with the PARP equivalent. Interestingly, upon doxycycline addition both constructs expressed similar quantities of tetR (1.3-fold higher for pTbFIX). This suggests that the unrepressed inducible rRNA promoter is capable of driving transcription of all three downstream genes ( Fig 2B).
In order to determine their regulation properties, transgenic cell lines were grown with different doxycycline concentrations. Western blot analysis showed that both vectors produced similar induction patterns. Abundance of the GFP reporter increased in a dose dependent manner reaching maximum levels with inducer concentrations greater than 50 ng/mL ( Fig  2C) similar to that reported for other inducible systems [17,21,38]. In these conditions, band quantification indicated~250 and~350 fold increase of gene expression in cells transfected respectively with pTbFIX and pTbFIX-PARP.
To confirm these findings and additionally to assess population homogeneity, samples were analyzed by flow cytometry (Fig 2D). In both cases, fluorescence histograms of uninduced transgenic cells overlapped that of the parental wild-type strain indicating tight repression of GFP transcription while addition of doxycycline to 1 μg/mL conduced to a uniform fluorescence increase. These properties were retained even after three months of continuous uninduced culture. In fact, with doxycycline concentrations above 50 ng/mL at least 95% of the cells exhibited fluorescence signal indicating a homogeneous population response (S2 Fig). Within intermediate concentrations the main differences between both cell lines became evident. In the presence of inducer concentrations ranging from 1 to 10 ng/mL, fewer parasites transfected with pTbFIX displayed comparatively lower GFP fluorescence than those transfected with pTbFIX-PARP. This was in agreement with the higher tetR abundance found in the former.
Finally, as can be seen in Fig 2E doxycycline removal can be used to repress transgene expression in these cell lines. In fact GFP abundances decline and reach undetectable levels after four days of culture in inducer free medium. Nevertheless, the fully induced state can be recovered by doxycycline readdition.

Inducible GFP expression in BSF
Transfections with both plasmids were performed on wild-type EATRO 1125 strain BSF trypomastigotes. All attempts involving pTbFIX-PARP were unsuccessful, probably due to the downregulation of the procyclin promoter and thus lower expression levels of the puromycin resistance gene. In contrast, viable clones were easily obtained with pTbFIX. Three independent clones were chosen in order to evaluate possible differences in expression patterns and regulation properties. Basal GFP expression levels in all three cell lines were close to undetectable ( Fig 3A). However, reporter induction already became evident after six hours of growth with 1 μg/mL of doxycycline. Maximum GFP abundances were reached 24 hours post  (Fig 3B). No significant deviations of the doubling times were found between induced (~6.4±0.4 h), uninduced (~6.8±0.3 h) and the wild-type parental strain (~6.5±0.5 h) (S3 Fig). However, at this point differences between the clones were already noticed with fold induction levels ranging 170 (clone A3) and~400 (clone D2). On the other hand, tetR abundances in the three parasite lines were equivalent and depending on doxycycline presence 1.5-fold or 2.1-fold higher than those in the well-established 90:13 strain (Fig 3C). These results suggested that the upstream inducible rRNA promoter also could contribute to repressor expression in BSF cells.
In this cellular context the system was very sensitive to inducer concentration reaching maximum expression with 10 ng/mL of doxycycline ( Fig 3D). On one hand, flow cytometry of these cell lines confirmed tight repression since fluorescence histograms of transgenic uninduced clones overlapped that of the parental wild-type strain ( Fig 3E). However, it also revealed additional differences between the clones. Fluorescence of clone A3 was less than that of clone D2. For its part, clone B6 contained the parasites showing the highest fluorescence. Similar to what was found for PCF, induction in the three clones depended on the presence of doxycycline in the medium. Two successive passages in inducer free conditions were enough to fully repress GFP expression, while doxycycline readdition was capable of restoring former expression levels ( Fig 3F).
Finally, to asses construct stability these cell lines were kept in culture in an uninduced state for three months with three weekly passages. Induction with different doxycycline concentrations was later analysed by flow cytometry (S4 Fig). Tight repression was retained in all three clones and maximal induction continued to be achieved with doxycycline concentrations above 10 ng/mL. No significant differences were found in population composition of clones A3 and D2, however clone B6 now contained equal amounts of responsive and unresponsive cells.

System performance in RNAi gene silencing
Inducible gene expression systems are frequently adapted for RNA interference mediated endogenous gene downregulation. To test if the vectors developed in this work could be used for this methodology both pTbFIX and pTbFIX-PARP were modified. The GFP coding sequence was replaced with two α-tubulin gene derived segments inserted in opposite orientations. Thus, transcription driven by the inducible rRNA promoter would produce a double stranded RNA molecule in a "stem-loop" configuration. The resulting plasmids (pTbFIX-αT and pTbFIX-PARP-αT) were transfected into Lister 427 PCF cells. No particular difficulties were experienced during the selection period and doubling times of the resulting transgenic lines (12.9±2.5 h and 11.2±0.7 h for pTbFIX-αT and pTbFIX-PARP-αT respectively) were comparable to that of the wild-type parental strain (12.9±0.8 h). However, upon doxycycline addition growth rates declined rapidly indicating impaired cellular viability. The effect was significant with both plasmids but more apparent in cells transfected with pTbFIX-αT 48 hours post induction (Fig 4A). Microscopic examination revealed the reported "FAT" phenotype [39] associated with α-tubulin downregulation in at least 90% of the cells in both cultures ( Fig  4B). 50000 events were captured. Mean fluorescence intensity values can be found in S1 Table. E) PCF cultures were diluted to 1x10 6 cells/mL and induced with 1 μg/mL of DOX. After 48 h parasites were extensively washed with DOX free medium and incubated for additionally 96 h, followed by DOX re-addition. GFP expression levels were monitored daily by Western blot analysis in total cell extracts (4x10 6 cells per lane). Tubulin was used as a loading control. Cultures were diluted every two days to 1x10 6 /mL to maintain cell density within a range that supports exponential growth. Regarding BSF, all attempts to transfect pTbFIX-αT into EATRO 1125 parasites were unsuccessful. This limitation might be explained by the already reported sensitivity of BSF even to the slightest basal downregulation of α-tubulin genes [40,41]. Alternatively, to test if the system could still be used for RNAi silencing of essential genes, two additional pTbFIX derivatives were obtained. With the same RNA "stem-loop" configuration pTbFIX-ENO and pTbFIX-CLH were constructed to target enolase and clathrin heavy chain genes respectively. Given the clonal variability in GFP expression patterns observed for pTbFIX, clones obtained after these transfections were initially screened for loss of viability associated with RNAi induction. In the case of pTbFIX-ENO, no proliferation could be detected in three out of four clones tested with the addition of 1 μg/mL of doxycycline. Two of these cell lines were chosen for further analysis. As can be seen in Fig 5A no growth rate defects could be detected in uninduced conditions. However, 24 hours after RNAi induction proliferation starts to decline and by 48 hours almost no further growth could be detected. Western blot analysis showed differences between the clones regarding enolase abundance (Fig 5B). In uninduced conditions, respective enzyme amounts in clones A1 and A3 were 78% and 93% that of the wild-type parental strain, suggesting tighter RNAi repression in the latter (Fig 5C). After 48 hours of induction values drop to 37% and 19% respectively, explaining the milder growth defects of clone A1.
On the other hand, the initial analysis of two clones obtained for pTbFIX-CLH showed that only one experienced loss of viability when cultured with doxycycline ( Fig 5D). Again no effect in proliferation were observed in uninduced conditions, however RNAi induction had even more severe growth defects than those obtained with pTbFIX-ENO. Furthermore, the reported cells per lane). For comparative purposes Lister 427 90:13 parasites were included in the analysis. D) Doxycycline dose-dependent GFP expression in BSF cells. Parasites were diluted to 3x10 4

cells/mL and induced for 48 h with indicated concentrations of DOX (clone B6). E) Fixed induced (DOX) or non-induced cells (NI)
were analysed by flow cytometry. Approximately 50000 events were captured. Mean fluorescence intensity values can be found in S2 Table. E) BSF cultures were diluted to 2x10 4 cells/mL and induced with 1 μg/mL of DOX. After 48 h parasites were diluted in DOX free medium and incubated for additionally 5 days, followed by DOX readdition (day 8). GFP expression levels were monitored by Western blot analysis in total cell extracts.

Discussion
This work was focused on the development of a simple genetic manipulation tool that would allow easy implementation of regulated transgene expression in Trypanosoma brucei. The main objective consisted in avoiding the dependence on pre-established cell lines, thus easily extending the range of strains on which to apply these methodologies. This could be accomplished with a single DNA molecule containing the sequences of interest as well as those that code for the required exogenous regulatory elements. To keep the design simple, the system was based exclusively on endogenous transcription activities and a minimum set of regulatory components. This was supported by previous works showing that T7 RNA polymerase is not a prerequisite for efficient inducible transgene expression in T. brucei [24,43]. Besides, apart from the PAC gene required for selection, the only other exogenous coded element was tetR. The arrangement of inducible and constitutive transcription units in pTbFIX was similar to that of other dual promoter systems [33]. Based on reported poor regulation properties, this configuration was later abandoned in favour of back-to-back alternatives [17]. However, unlike our design, in these pioneering works tetR was expressed from other genomic loci and did not contain a nuclear localization signal which is a feature included in more recent developments [19,20,23]. Indeed, the tandem "head-to-tail" configuration was chosen considering that eventual unregulated transcription by the leftmost rRNA promoter in pTbFIX would contribute to tetR expression in a way reminiscent of a negative feedback mechanism. The increase in repressor abundance upon doxycycline addition, most evident in PCF transfected with pTbFIX-PARP, supports this possibility. Finally, during the design phase particular care was taken in order to minimize alterations to the physiological transcription patterns of the cells. In this sense, promoters in pTbFIX vectors and those of the targeted rRNA loci share the same orientation after construct integration.
Transgenic PCF cells experienced equally tight regulation of GFP expression with pTbFIX and pTbFIX-PARP, indicating that tetR levels produced by the former were significantly above those required to repress rRNA transcription. Consequently, further system improvements should be focused on other aspects than increasing repressor amounts. On the other hand, in the fully induced state both constructs reached similar GFP expression levels and cell populations were notably homogeneous. For practical purposes both vectors are equally well suited for conducting regulatable transgene expression based methodologies in PCF cells. However, pTbFIX corresponded to the most versatile tool since it can also be used in BSF. Regulation properties in this cellular background were similar to those observed in PCF. The most apparent difference related to clonal variability, which has already been reported for other constructs [32,33]. The fact that transgene induction and repression are reversible in all these PCF and BSF lines implies that the system developed can be employed in conditional knockout methodologies.
In principle, pTbFIX has good construct stability properties similar to those reported for other rRNA loci targeted molecules [44]. After three months of continuous culture both PCF cell lines exhibited almost no change in regulation properties and population composition. For their part only one of the three BSF clones experienced loss of GFP expression in a fraction of the cells and, most importantly, all clones retained high repression in uninduced conditions.
It can be speculated that soon after pTbFIX integrates into the genome some unregulated transgene expression might take place until repressor abundances increase. Yet, even with this delay in repression, it was still possible to obtain viable cell lines with constructs for the RNAi downregulation of essential genes in both PCF and BSF parasites. Interestingly, while in uninduced PCF populations obtained with both pTbFIX-αT vectors no cells were found with altered morphology, they showed a noticeably homogeneous response towards induction of RNAi. The former could be due to rapid counter selection of cells with loose repression.
The BSF clonal variability observed not only for GFP expression constructs but also for those intended for RNAi downregulation of enolase as well, can be explained by the already reported position effects associated to alternative site of insertion among different clones [32,33,45]. In this respect, besides their use as general purpose transgene expression tools, the pTbFIX vectors might be employed to perform comparative integration site studies in different strains.
Finally, all RNAi constructs in this work were obtained with conventional restriction enzyme based techniques, since these correspond to the most widely available alternative. Nevertheless, pTbFIX vectors can easily be made compatible with other cloning technologies and thus more amenable for high-throughput methodologies [46][47][48].  Table. (TIFF) S1