Transient and selectable transformation of the parasitic protist  Trichomonas vaginalis

Abstract

We have developed methods to transiently and selectably transform the human-infective protist Trichomonas vaginalis. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into T. vaginalis by electroporation and have used the 5 and 3 untranslated regions of the endogenous gene ␣-succinyl CoA synthetase B (␣-SCSB) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 g closedcircular construct. Optimal levels of expression were observed using Ϸ2.5 ؋ 10 8 T. vaginalis cells and 350 volts, 960 Fd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in T. vaginalis transient transformants revealed the highest level of expression 8-21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (neo) gene as the selectable marker. Cells electroporated with 20 g of the NEO construct were plated in the presence of 50 g͞ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3-5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10-30 copies per cell. The ability to transiently and selectably transform T. vaginalis should greatly enhance research on this important human parasite.

Trichomonas vaginalis Hmp35, a Putative Pore-forming Hydrogenosomal Membrane Protein, Can Form a Complex in Yeast Mitochondria

“…For generation of the pHmp35H plasmid, we used the previously described (18) plasmid construct pHmp31-(HA) 2 which carries a neomycin phosphotransferase (neo) cassette that allows selection of transformants in T. vaginalis (25). The hmp35 open reading frame was amplified from the genomic clone MP40.1 using the forward primer Nhmp35H (Table I) to introduce an NdeI restriction site at the 5Ј end of the PCR product and the reverse primer BAhmp35H (Table I) to introduce a hexahistidine codon at the 3Ј end, followed by a BamHI restriction site.…”

“…Selectable Transformation of T. vaginalis-T. vaginalis T1 cells were electroporated with 50 g of pHmp35H as described previously (25), and transformants (35H) harboring the plasmid were selected with 200 g/ml geneticin (Invitrogen).…”

“…This fusion protein is expressed from an episomal plasmid, pHmp35H (Fig. 6A), which also contains the neomycin phosphotransferase gene that confers resistance to the drug geneticin and provides for selection (18,25). The Hmp35-His 6 protein was efficiently targeted to the organellar pellet as shown by a crude cell fractionation and Western analysis with an anti-hexahistidine antibody (Fig.…”

Presence of a Member of the Mitochondrial Carrier Family in Hydrogenosomes: Conservation of Membrane-Targeting Pathways between Hydrogenosomes and Mitochondria

“…The ␣-SCSB-CAT (␣-succinyl coenzyme A synthetase B-chloramphenicol transferase) construct described previously (8) was modified by introducing an NdeI restriction site at the initiation codon of the ␣-SCSB gene by site-directed mutagenesis. All primers used are listed in Table 1.…”

“…To study the role of the Hmp31 presequence in translocation, we have expressed mutant and chimeric hydrogenosomal proteins in T. vaginalis (8,27) using the influenza HA epitope to detect the proteins. Constructs carrying the HA-tagged genes also carry a neo gene cassette (24) for selection of transformants.…”