ABSTRACT
Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.
Subject(s)
Entamoeba histolytica/metabolism , Protozoan Proteins/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Blotting, Western , Cell Nucleus/chemistry , Computational Biology , Cytoplasm/chemistry , Electrophoretic Mobility Shift Assay , Entamoeba histolytica/chemistry , Entamoeba histolytica/genetics , Fluorescent Antibody Technique, Indirect , Gene Expression Profiling , Humans , Microscopy, Electron, Transmission , Protein Binding , Protozoan Proteins/genetics , Sequence Homology, Amino Acid , Telomere-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
Cysteine Endopeptidases/metabolism , Cysteine Proteases/metabolism , Protozoan Proteins/metabolism , Trichomonas vaginalis/enzymology , Cell Adhesion , Cysteine Proteases/chemistry , Cysteine Proteases/physiology , Female , Golgi Apparatus/metabolism , HeLa Cells , Humans , Immunoglobulin G/immunology , Iron/metabolism , Liver/parasitology , Phosphorylation , Protozoan Proteins/chemistry , Protozoan Proteins/physiology , Recombinant Proteins/metabolism , Trichomonas Vaginitis/parasitology , Trichomonas vaginalis/pathogenicity , Vagina/parasitology , Virulence Factors/metabolismABSTRACT
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.
