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, diarrheacausing 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.

Hydrogenosomes in the diplomonad Spironucleus salmonicida

“…S14). Five of the candidate proteins, derived from all three categories, were found to display a subcellular localization consistent with endoplasmic reticulum in S. salmonicida 8. One of the chosen proteins of the hydrogenosome-enriched fraction, a divergent paralogue of acetyl-CoA synthetase (ADP-forming), was found to display a hydrogenosomal localization (Fig.…”

“…We utilized our draft genome sequence of the salmon parasite S. salmonicida in combination with the recently developed stable transfection system for the parasite8 to investigate the nature of its MROs and the origin, extent and basis for hydrogen production in Spironucleus . We constructed a S. salmonicida strain that carries a chromosomally integrated haemagglutinin (HA)-tagged Chaperonin 60 (Cpn60), a marker for MROs, and employed immunofluorescence to investigate its subcellular localization.…”

“…S. salmonicida (ATCC 50377, previously ATCC 50380), S. barkhanus 26 and S. vortens (ATCC 50386) were obtained from the American Type Culture Collection (ATCC) and grown in axenic cultures in modified LYI culture media8 at 16, 4 and 22 °C, respectively. G. intestinalis isolate WB clone C6 (ATCC 50803) was cultivated in TYDK media at 37 °C in 10 ml tightly capped slanted culture tubes (NUNC)27.…”

“…Transfection and maintenance of S. salmonicida transfectants followed the methods described in Jerlström-Hultqvist et al 8. Briefly, one confluent tube of S. salmonicida cells (~10 6  cells per ml) was placed in ice slush 15 min and repeatedly tapped to detach the cells.…”

Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia

“…3c)52. Both constructs carry CWP1 (CWP1-BSR-pBS) or C-terminally HA tagged CWP2 (CWP2HA-BSR-pBS) open reading frames driven by a minimal CWP1 promoter.…”