Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

Abstract

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

Figure 1. Putrescine effect on the TvCP39 activity from T. vaginalis.

A) Putrescine effect on the proteolytic activity of T. vaginalis. Zimograms using total proteinases from parasites grown in normal media (N)(lane 1), DAB-treated parasites (D)(lane 2), DAB-treated parasites transferred into exogenous putrescine (DP)(lane 3), DAB-treated trichomonads transferred into a normal medium (DN)(lane 4) and parasites grown in normal medium transferred into an exogenous putrescine media (NP)(lane 5). B) Polyamine effect on the proteinases activity bound to HeLa cells. Ligand-proteinases assays using untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated parasites transferred into exogenous putrescine media (DP)(lane 3), DAB-treated parasites transferred into normal media (DN)(lane 4) and parasites grown in normal media and transferred into an exogenous putrescine media (NP)(lane 5). Arrowhead shows the TvCP30 proteolytic activity. C) Densitometry analyses of TvCP39 proteolytic activity bands from panel B. Bars indicate the average of the intensity of TvCP39 activity bands from three independent ligand-proteinases assays and error bars represent the standard deviations.

Figure 2. Putrescine effect on TvCP39 transcript and protein.

A) Semi-quantitative RT-PCR analysis performed with total RNA from untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2,); DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP)(lane 3); DAB-treated trichomonads transferred to normal medium (DN)(lane 4), and trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP)(lane 5) to amplify 238 bp from the tvcp39. A 112 pb amplicon from β-tubulin was amplify as a loading control. B) qRT-PCR of samples described in A. The Ct levels of tvcp39 mRNA in trichomonads after DAB treatment (bar D) decreased at 20% but the tvcp39 mRNA were restored (70%) by adding 40 mM exogenous putrescine to DAB-treated parasites (bar DP). C) Total protein extract from T. vaginalis grown in normal media (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated trichomonads transferred into exogenous putrescine media (DP)(lane 3); DAB-treated parasites transferred into normal medium (DN)(lane 4) and trichomonads grown in normal medium transferred to medium with 40 mM exogenous putrescine (NP)(lane 5) were blotted onto nitrocellulose membranes and incubated with anti-TvCP39 and anti-α-tubulin (loading control) antibodies. Arrowheads indicate the immunodetected protein for each antibody employed.

Figure 3. Putrescine effect on TvCP39 localization.

A) TvCP39 localization in the polyamine presence. Immunofluorescence analysis of fixed, permeabilized (P; 1–4, 9–12, and 17–20) and Non permeabilized (NP; 5–8, 13–16, and 21,24) parasites untreated (N) (1–8), DAB-treated (D) (9–16), or DAB-treated transferred into exogenous putrescine media (DP) (17–24) incubated with the anti-TvCP39 antibody (1–24) or preimmune sera (PI; 25–28) followed by secondary anti-mouse conjugated to a fluorescein isothiocyanate (Jackson) antibody (1∶90 dilution) and mounted with Vectashield-DAPI. Photographs were taken under laser confocal microscopy (Leica, DMLS). B) Re-localization of TvCP39. Immunofluorescence analyses of fixed and permeabilized parasites that were untreated (Panel N1 to N6) or DAB-treated (Panel D1 to D6), or DAB-treated transferred into exogenous putrescine media (Panel DP1 to DP6), or normal culture parasites that were transferred into exogenous putrescine media (Panel NP1 to NP6). The parasites were incubated with the antibody raised against TvCP39 (green) and anti-HSP70 (red) or with preimmune sera (Panel PI). Nuclei are labeled with DAPI (blue). Photographs were taken under laser confocal microscopy (Leica, DMLS). Scale bar = 10 µm.

Figure 4. TvCP39 re-localization after DAB treatment and putrescine restoration.

A) Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4) were blotted into a nitrocellulose membrane and incubated with anti-TvCP39, anti-TveIF-5A (control of cytoplasmic protein), anti-nucleoporin (control of nuclear protein) and anti-PCNA (control of nuclear protein) antibodies. Arrowheads show TvCP39 (39 kDa), the TveIF-5A (20 kDa), the nucleoporin (53 kDa), and the PCNA (28 kDa) protein bands. B) Zymograms from Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4). Arrowhead indicates the TvCP39 proteolytic activity.

Figure 5. The tvcp39 mRNA and protein stabilities are regulated by putrescine.

A) RNAm levels of tvcp39 by semi-quantitative RT-PCR analysis using total RNA from parasites treated with actinomycin D and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP); or DAB-treated trichomonads transferred to normal medium (DN); or trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP). Samples were taken at 0, 1, 3, 6, 8, 12 and 24 h for amplification of 110 pb of tvcp39 mRNA and 112 bp of β-tubulin mRNA (β-tub)(loading control). Arrowheads indicate the amplification products obtained. B) Transcriptional blockade using actinomycin D. Trichomonads grown in normal medium (N), DAB-treated trichomonads (D), DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP), DAB-treated trichomonads transferred into normal medium (DN), and trichomonads grown in normal medium and transferred into an exogenous putrescine medium (NP) were treated with actinomycin D. Samples taken at several times (0, 1, 2, 6, 8, 12, and 24 h) were use to amplified the tvcp39 mRNA which was quantified by densitometric analysis and normalized. Bars represent the mean of each sample and the standard errors were included. C) Blockage of protein synthesis by cycloheximide. Trichomonads were treated with 10 µg of cycloheximide and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP). Samples were taken at several times for Western blot analysis using anti-TvCP39 (dilution 1: 1000) and α-tubulin (dilution 1∶100) antibodies. Arrowheads indicate the TvCP39 and α-tubulin proteins. D) Densitometric analysis of the samples described in C. The bands corresponded to TvCP39 were quantified and normalized to α tubulin. Bars represent the mean of three biological triplicates.