Isolation, Partial Purification, and Immunogenicity of Flagella from Tritrichomonas foetus

Parasite cell culture and isolation of flagella. Tritrichomonas foetus, strain 330.13, was cultured axenically at 37°C in Diamond's trypticase-yeast extract-maltose (TYM) medium, pH 7.2, supplemented with 10% heat-inactivated horse serum (Diamond 1957). Gentamicin and fungizone were added at 20 and 0.25 μg/ml to control bacterial and fungal contamination, respectively. Two-liter cultures were harvested at mid-logarithmic phase and used for each flagellar preparation experiment. Flagella of T. foetus were isolated essentially as previously described (Jemilohun 1998) with some modifications, using calcium or mastoparan deflagellation buffer protocols (Quarmby and Hartzell 1994; Sanders and Salisbury 1989). The cells were washed with PBS buffer and suspended in 20 ml of deflagellation buffer (DB) containing 0.25 M sucrose, 20 mM HEPES, (pH 7.2), 2 mM MgCl₂, 15 mM CaCl₂, and 1 mM 2-mercaptoethanol. Phenylmethylsulfonyll fluoride (PMSF) and Na-p-tosyl-l-lysine chloro-methyl ketone (TLCK) were added to final concentrations of 3 and 5 mM, respectively. The cell suspension was deflagellated by vortex homogenization for 3 min. Cell lysis was minimal and about 95% deflagellation was achieved (data not shown). Deflagellation was monitored by phase contrast microscopy.

The homogenate was diluted 10 times with DB and centrifuged at 153 g for 10 min to remove the deflagellated cells. The supernatant containing the flagella was collected and the pellet was resuspended in fresh buffer, diluted, and centrifuged as before. The supernatant was collected and added to the first. The combined supernatant was centrifuged at 153 g for 10 min and the resulting supernatant was centrifuged at 9,820 g for 20 min to pellet the flagella. The flagellar pellet was suspended in 40-ml DB and centrifuged at 153 g for 10 min to remove sub-cellular contaminants. The supernatant was carefully aspirated into another centrifuge tube and centrifuged at 9,820 g for 20 min. The resulting pellet was suspended in 2-ml buffer and termed the “crude flagellar prep.” Observation by phase-contrast microscopy showed that the crude flagellar prep was enriched with flagella and without intact cell contaminants. The crude flagellar preparation was further purified by a 1–1.8 M linear sucrose gradient centrifugation (Opperdoes et al. 1984) and percoll discontinuous centrifugation (Morgado Diaz and De Souza 1997). Both sucrose and percoll gradients were centrifuged at 22,600 g for 2 h in a Beckman J2-HS centrifuge using JA 13.1 rotor (Beckman Instruments Inc., Palo Alto, CA). The bands containing the flagella were carefully removed, diluted with DB, and centrifuged at 9,820 g for 20 min. The flagellar pellet was carefully suspended in DB with PMSF and TLCK added to 3 mM final concentration and stored at −20°C until use.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The flagellar preparations and the whole-cell sample were solubilized in buffer consisting of 50 mM Tris-HCl (pH 8.2), 5 mM EDTA, 1% (v/v) NP-40, 0.1% (w/v) SDS, 3 mM TLCK, and 1 mM PMSF. Protein concentration was determined by the bicinchoninic acid (BCA) method using a commercial kit (Pierce, Rockford, IL) (Smith et al. 1985). Bovine serum albumin was used as the protein standard. Flagellar preparations yield an average of 17 mg of flagellar proteins. SDS-PAGE was performed in a mini-gel apparatus (Bio-Rad Laboratories, Hercules, CA) with 10% separating polyacrylamide gel and 5% stacking system. The samples were solubilized under denaturing conditions in 5 × sample buffer (20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 4% (w/v) SDS, 125 mM Tris-HCl, pH 6.8). The samples were heated at boiling for 5 min prior to loading equal amounts of proteins (15 μg) to the wells of the gel. Broad-range molecular mass standards of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa (Bio-Rad Laboratories, Hercules, CA) or molecular mass ladders, 220, 160, 120, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, and 10 kDa (Invitrogen, Carlsbad, CA) were used. Protein separation was accomplished at 200 constant volts (Laemmli 1970). To visualize protein bands, gels were stained with 0.1% (w/v) Coomassie Blue R-250 in solution containing 40% (v/v) methanol and 10% (v/v) acetic acid. The gels were destained in solution containing 40% methanol and 10% acetic acid (Bio-Rad Laboratories).

Antibodies and immunoblot analyses. To identify flagellar immunogens, flagellar prep and whole-cell samples were analyzed by immunoblots. The blots were probed with bovine antisera, rabbit antisera, and a monoclonal antibody kindly provided by Drs. Donald E. Burgess (ATTC, Manassas, VA) and Lynette B. Corbeil (UC, San Diego, CA). The bovine antibodies were prepared by immunizing cattle intramuscularly twice at biweekly intervals with 10⁷T. foetus cells in 1% formalinized saline mixed with incomplete Freund's adjuvant. Serum was collected 10 d after the last immunization (Corbeil et al. 1989). The monoclonal antibodies were raised by intraperitoneal injection of mice with approximately 10⁷T. foetus cells mixed with complete Freund's adjuvant. Mice were boosted by intravenous injection with T. foetus 3 d prior to fusion. Positive clones were selected and hybridoma clones were prepared by limiting dilution (Burgess 1986). Rabbit antibodies were prepared by immunizing rabbits with 10⁶T. foetus cells mixed with an equal vol. of Freund's complete adjuvant. The immunizations were repeated with Freund's incomplete adjuvant (Corbeil, Hodson, and Widders 1991; Shaia et al. 1998).

After fractionation of the samples by SDS-PAGE, the proteins were electroblotted to nitrocellulose membrane for 1.5 h at 100 V (Towbin, Staeehelin, and Gordon 1979). The membranes were washed briefly in PBS buffer and the non-specific binding sites of the nitrocellulose membranes was blocked at room temp. for 2 h in a blocking buffer (5% (w/v) blotto, 0.2% (v/v) Tween 20 in PBS). The membranes were briefly rinsed with 0.2% Tween 20 in PBS (PBST buffer) and probed overnight with 1:250 dilution bovine antisera (Corbiel et al. 1989), 1:250 dilution of rabbit antisera (Corbiel et al. 1991), 1:100 dilution monoclonal antibody (MAb) (Burgess 1986), and 1:200 dilution of rabbit antibodies (Shaia et al. 1998) prepared in PBST containing 1% blotto at 4°C with gentle shaking. The membranes were washed three times for 10 min each with PBST and bound antisera were detected by incubation for 30 min at room temp. in anti-bovine or anti-rabbit or anti-mouse IgG-alkaline phosphatase conjugate diluted with 1% blotto in PBST according to the supplier specifications (Kirkegaard and Perry Laboratories Inc., Gaiththersburg, MD). The blots were washed three times with 1% blotto in PBST and once with PBS and developed with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as the substrate and nitroblue tetrazolium (NBT) as the chromogenic indicator (Kirkegaard and Perry Laboratories Inc.). The reaction was stopped by washing the blots in distilled water.

Microscopic analyses. Although some of the parasite cells were broken in the process of deflagellation, which led to the presence of subcellular particles, these unwanted contaminants were removed by low speed differential centrifugation and linear sucrose gradient centrifugation. The flagellar preparation was about 85–95% pure based on analysis by phase contrast microscopy (Fig. 1). Further purification of the flagellar fraction was achieved by linear sucrose gradient or percoll gradient centrifugation. The flagellar sheath (membrane) remained intact and was not affected by the method of deflagellation nor the purification scheme employed (Fig. 2). Flagellar complexes (C), flagellar fragmentation (F), and flagellar spherical formation (S) were apparent (Fig. 2).

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Highly reproducible gel patterns were obtained from analyses of several different flagellar preparations. The whole flagella showed a complex pattern of polypeptides ranging from about 10 to 300 kDa (Fig. 3). Three prominent proteins 42, 49, and >250 kDa, and several minor proteins were consistently observed in the partially purified flagellar preps. These three prominent proteins were enriched in the flagellar preparation compared to the whole-cell extract, suggesting that they are important flagellar proteins (Fig. 3).

Immunoblot analyses. The bovine antisera demonstrated immunoreactivity mainly with high molecular mass antigens of about 100, 116, and 180 kDa from both whole-cell and flagellar extracts. The 116-kDa antigen was uniquely located on the flagella as its band-intensity on the whole cell extract was reduced (Fig. 4A, lane 2). The MAb showed strong immunoreactivity with an 84-kDa protein and other higher molecular mass antigens, suggesting that these antigens share some epitopes with the 84-kDa antigen. Along with several other antigens, the 84-kDa antigen was strongly recognized in both whole-cell and flagellar samples (Fig. 4B). The two heterologous rabbit antisera showed broad-spectrum immunoreactivity with flagellar extract (Fig. 5), while the other rabbit antisera reacted essentially with high molecular mass antigens (Fig. 5B).