Stable transfection of the diplomonad parasite Spironucleus salmonicida

Abstract

Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II-FLAG tandem-affinity purification tag or streptavidin binding peptide-glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus.

Fig 1

Schematic pSpiro vector maps showing constructed cassettes and relevant restriction sites. The episomal vectors contain the ampicillin resistance gene (AmpR) for selection in E. coli and the pac, bsr, and nptII genes for selection in S. salmonicida. The pac and bsr genes are flanked by the 5′ UTR and 3′ UTR of the ornithine carbamoyltransferase (OCT) gene, which are cloned between the HindIII/NcoI and XhoI/KpnI sites, respectively. The nptII gene is inserted between the 5′ UTR and 3′ UTR of the fructose bisphosphate aldolase (FBPA) gene, and the entire cassette is cloned between SpeI and NdeI. The three eukaryotic selection markers can be exchanged using combinations of KpnI/SpeI and NdeI/HindIII. The 3′UTR from the ribosomal protein S15A (RbpS15A) gene was cloned between the ApaI and SacI sites. (A) The C-terminal cassettes (3×HA, 2×OLLAS, 3×MYC, SBP-GST, and SF-TAP) are cloned between the NotI and ApaI sites. Ten unique restriction sites are available for cloning in the MCS region of the pSpiro C-terminal tagging vectors. (B) The N-terminal vectors carry the α-tubulin (αTub) 5′ UTR cloned between the HindIII and EcoRV sites. The N-terminal tagging cassettes (3×HA, 2×OLLAS, SBP-GST, and SF-TAP) are cloned at the EcoRV and BamHI sites. Six restriction sites are available for cloning in the MCS region of the pSpiro N-terminal tagging vectors.

Fig 2

Expression and localization of epitope-tagged proteins in S. salmonicida. Stably transfected S. salmonicida carrying the pSpiro-PAC-fibrillarin-3×HA and pSpiro-PAC-caltractin-3×HA episomal plasmids were fixed using 2% PFA and methanol and acetone, respectively. (A) Fixed cells were blocked with 2% BSA; they were stained using rabbit anti-HA (1:1,600) and anti-tubulin TAT1 (1:150) and detected using anti-rabbit Alexa Fluor 594 (A594) (1:250) and anti-mouse Alexa Fluor 488 (A488) (1:200), respectively. (B) Cells were stained with mouse monoclonal anti-centrin 20H5 (1:100) and rabbit anti-HA (1:1,600) and detected by anti-mouse A488 and anti-rabbit A594, respectively. The cells were mounted in VectaShield medium containing DAPI and viewed using a Zeiss 510 laser scanning confocal microscope. (A and B, upper left) Maximum intensity projection (MIP) of the A594 signal (red). (A, upper right) MIP of the A594 (red) and DAPI (blue) signals. (B, upper right) MIP of A488 (green). (A and B, lower left) MIP of A488 (green), A594 (red), and DAPI (blue). (A and B, lower right) Bright-field image from the center of the confocal Z-stacks. Fibrillarin-3×HA displays nuclear localization showing partial colocalization with DAPI but enrichment in the regions devoid of DAPI stain. Caltractin-3×HA shows colocalization to the basal body marker centrin in the triangular foci anterior to the nuclei. Scale bars, 5 μm. (C) Western blot of S. salmonicida cells using the mouse monoclonal anti-centrin (20H5) antibody. A doublet of bands is observed at ∼20 kDa. (D) Western blot of S. salmonicida transfectants expressing HA-tagged proteins using the mouse monoclonal anti-HA antibody. From left to right, shown are IFT46-3×HA transfectants (theoretical molecular mass, 30.4 kDa), caltractin-3×HA transfectants (23.4 kDa), fibrillarin-3×HA transfectants (39.1 kDa), and S. salmonicida wild-type cells. (E) Western blot of S. salmonicida transfectants expressing OLLAS-tagged proteins using the rabbit anti-OLLAS antibody. From left to right, shown are S. salmonicida wild-type cells, BiP-2×OLLAS transfectants (75.1 kDa), PDI2-2×OLLAS transfectants (44.5 kDa), and Sec61α-2×OLLAS transfectants (55.6 kDa).

Fig 3

Localization of 3×HA epitope-tagged IFT46 S. salmonicida trophozoites. Transfectants carrying the IFT46-3×HA construct were fixed in 2% PFA and blocked with 2% BSA; they were stained using rabbit anti-HA (1:1,600) and anti-tubulin TAT1 (1:150) and detected using anti-rabbit Alexa Fluor 594 (A594) (1:250) and anti-mouse Alexa Fluor 488 (A488) (1:200), respectively. The cells were mounted in VectaShield medium containing DAPI and viewed using a Zeiss 510 laser scanning confocal microscope. (A) Maximum intensity projection (MIP) of A594 (red) and DAPI (blue) signals; (B) MIP of A488 (green) and DAPI signals; (C) MIP of A488, A594, and DAPI signals; (D) bright-field image from the center of the confocal Z-stack. IFT46-3×HA displays localization to paired cylindrical foci above the nuclei, to sheets that pass through the cell body, and to foci along the flagella sometimes being enriched at the tips. Diffuse staining of the cell body is commonly observed. Scale bars, 10 μm.

Fig 4

Localization of Sec61α-2×OLLAS, BiP-2×OLLAS, PDI2-2×OLLAS, 2×OLLAS, IFT46-3×HA, and PDI2-2×OLLAS in S. salmonicida trophozoites. Cells carrying constructs of Sec61α-2×OLLAS (A), BiP-2×OLLAS (B), PDI2-2×OLLAS (C), and IFT46-3×HA, PDI2-2×OLLAS (D) were fixed using 2% PFA and blocked using 2% BSA in PBS. (A to C) Cells were reacted using the anti-OLLAS rabbit (1:2,000) and anti-tubulin TAT1 (1:150) antibodies and detected using anti-rabbit antibody conjugated to Alexa Fluor 594 (A594) (1:250) and anti-mouse antibody conjugated to Alexa Fluor 488 (A488) (1:200), respectively. (D) Cells were incubated with anti-OLLAS rabbit (1:2,000) and mouse monoclonal anti-HA (1:500) and detected with anti-rabbit antibody conjugated to A594 (1:250) and anti-mouse antibody conjugated to A488 (1:200), respectively. The cells were mounted in VectaShield medium with DAPI and viewed using a Zeiss 510 laser scanning confocal microscope. (A to C, left panel) Maximum intensity projection (MIP) of A594 (red); (A to C, middle) MIP of DAPI (blue) and A488 (green) signals; (A to C, right) overlay of the left and middle panels. The three proteins in panels A to C localize to similar structures enveloping the nuclei and are distributed along the length of the recurrent flagella. (D, upper left) MIP of DAPI (blue) and A594 (red) signals; (D, upper right) MIP of DAPI (blue) and A488 (red) signals. The lower-left panel shows an overlay of the upper panels. The lower-right panel shows the cells in bright-field view. (D) The cells display labeling of foci on the flagella, paired cylindrical signals above the nuclei, two sheets that run the length of the cell to posterior end, and cytoplasmatic staining in the green channel and labeling around the nuclei and along the recurrent flagella in the red channel. Scale bar, 5 μm.