A high-throughput cloning system for reverse genetics in Trypanosoma cruzi

Abstract

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

Figure 1

Southern blot analysis of transfected T. cruzi cells. Lanes represent HindIII-digested: genomic DNA from T. cruzi wild type (WT), from T. cruzi transfected with the TAPneo-Tcpr29A plasmid (29A) and TAPneo-Tcpr29A isolated plasmid (Control). The neomycin resistance marker (NEO) and the tandem affinity purification tag (TAP) were used as probes. 1 Kb Plus DNA Ladder (Invitrogen) was used as the molecular weight marker.

Figure 2

Levels of GFP-fused and NEO recombinant mRNAs in T. cruzi. The Y-axis indicates the level of GFP and NEO mRNA quantified by real-time RT-PCR using populations of cells transfected with GFPneo-Rab7, GFPneo-PAR2 and GFPneo-CTRL.

Figure 3

Detection of GFP-fused recombinant proteins and FACS analysis. Lanes in A and B represent protein extracts from T. cruzi wild type (WT) cells and cells transfected with GFPneo-CTRL, GFPneo-Rab7 and GFPneo-PAR2. In A is represented the load control gel. In B, these extracts were incubated with antibodies against GFP. BenchMark (Invitrogen) was used as the molecular weight marker. In C, T. cruzi wild type epimastigotes (WT) were used as a negative control. For each culture, 20,000 cells were counted. The Y- and X-axis represent the number of cells counted (events) and GFP fluorescence (FL1-H) in arbitrary fluorescence units (AFU), respectively.

Figure 4

Subcellular localization of TcRab7 and PAR 2 in T. cruzi using pTcGW vectors. Fluorescence microscopy of epimastigotes transfected with GFPneo-CTRL, GFPneo-PAR2, GFPneo-Rab7, GFPhyg-PAR2 and CFPneo-Rab7. The merged frame was composed by "GFP" and "DAPI" images overlap. The DAPI frame in the last row was replaced by a frame containing the cyan fluorescence-Rab7 construct (*), in which a red signal was used. The "#" frame contains a merger of DAPI/GFPhyg-PAR2/CFPneo-Rab7.

Figure 5

Efficiency of L27 and 29A complexes purification with the original TAP tag tested in T. cruzi cells. In A, the TAP tag-fused TcrL27 (L27), Tcpr29A (29A) and the control TAPneo-CTRL (CTRL) was detected by western blot with anti-CBP antibody. In B, the fractions from TAP purification were probed with anti-L26 and anti-α2 in immunoblots. Lanes represent total protein (T) or eluted product after digestion (E). BenchMark (Invitrogen) was used as the molecular weight marker.

Figure 6

Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (Cm^R), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the vector reading frame with start and stop codons are shown.