RESUMO
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 â¼ 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.
Assuntos
Cisteína Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Putrescina/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Western Blotting , Divisão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cisteína Proteases/genética , Expressão Gênica/efeitos dos fármacos , Microscopia Confocal , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/genética , Putrescina/análogos & derivados , Putrescina/antagonistas & inibidores , Putrescina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trichomonas vaginalis/citologia , Trichomonas vaginalis/genéticaRESUMO
The causal agent of trichomoniasis is a parasitic protist, Trichomonas vaginalis, which is rich in proteolytic activity, primarily carried out by cysteine proteases (CPs). Some CPs are known virulence factors. T. vaginalis also possesses three genes encoding endogenous cystatin-like CP inhibitors. The aim of this study was to identify and characterize one of these CP inhibitors. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), a cystatin-like peptidase inhibitor dubbed Trichocystatin-2 (TC-2) was identified in the T. vaginalis active degradome in association with TvCP39, a 39kDa CP involved in cytotoxicity. To characterize the TC-2 inhibitor, we cloned and expressed the tvicp-2 gene, purified the recombinant protein (TC-2r), and produced a specific polyclonal antibody (α-TC-2r). This antibody recognized a 10kDa protein band by western blotting. An indirect immunofluorescence assay (IFA) and cell fractionation assays using the α-TC-2r antibody showed that TC-2 was localized in the cytoplasm and lysosomes and that it colocalized with TvCP39. TC-2r showed inhibitory activity against papain, cathepsin-L, and TvCP39 in trichomonad extracts and live parasites but not legumain-like CPs. Live trichomonads treated with TC-2r showed reduced trichomonal cytotoxicity to HeLa cell monolayers in a TC-2r-concentration-dependent manner. In this study, we identified and characterized an endogenous cystatin-like inhibitor in T. vaginalis, TC-2, which is associated with TvCP39 and appears to regulate the cellular damage caused by T. vaginalis.
Assuntos
Cistatinas/farmacologia , Cisteína Proteases/química , Inibidores de Proteases/farmacologia , Tricomoníase/tratamento farmacológico , Trichomonas vaginalis/enzimologia , Animais , Apoptose , Sequência de Bases , Western Blotting , Catepsina L/antagonistas & inibidores , Proliferação de Células , Células Cultivadas , Clonagem Molecular , Cistatinas/genética , Cistatinas/imunologia , Cisteína Endopeptidases/química , Cisteína Proteases/metabolismo , Citoplasma/enzimologia , Eletroforese em Gel Bidimensional , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Lisossomos/enzimologia , Masculino , Dados de Sequência Molecular , Filogenia , Inibidores de Proteases/imunologia , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Tricomoníase/metabolismo , Tricomoníase/microbiologia , Trichomonas vaginalis/genéticaRESUMO
The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.
Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/enzimologia , Adesão Celular , Cisteína Proteases/química , Cisteína Proteases/fisiologia , Feminino , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Imunoglobulina G/imunologia , Ferro/metabolismo , Fígado/parasitologia , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Proteínas Recombinantes/metabolismo , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/patogenicidade , Vagina/parasitologia , Fatores de Virulência/metabolismoRESUMO
TvCP39 is a 39 kDa cysteine proteinase (CP) involved in Trichomonas vaginalis cytotoxicity that has been found in vaginal secretions and is immunogenic in patients with trichomonosis. The goal of this work was to identify, clone, express, and characterize the tvcp39 gene. The tvcp39 gene was identified using a proteomic approach, and the complete gene was amplified using PCR, cloned, and sequenced. TvCP39 is encoded by a 915-bp cathepsin L-like CP gene. A fragment corresponding to the mature region (TvCP39r) was expressed, purified, and used to produce rabbit polyclonal antibodies and in functional assays. In one- and two-dimensional western blot assays, the anti-TvCP39r antibody reacted with two protein bands of ~28 and 27 kDa and three spots of ~28, 27, and 24 kDa in trichomonad proteinase-rich extracts that could correspond to the mature and processed fragments of the TvCP39 peptidase. The anti-TvCP39r antibody reacted with the parasitic surface and the native TvCP39 present in vaginal washes from patients with trichomonosis. Moreover, the recombinant TvCP39 protein bound to the surface of HeLa cells and protected HeLa cell monolayers from trichomonal destruction in a concentration-dependent manner. In conclusion, our data support TvCP39 as one of the surface proteinases that is glycosylated and is involved in trichomonal cytotoxicity. Thus, TvCP39 is the first glycosylated cysteine proteinase detected in T. vaginalis.