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Actin and tubulin proteins from Trichomonas vaginalis are crucial for morphogenesis and mitosis. This parasite has 10 and 11 genes coding bonafide actin and tubulin proteins, respectively. Hence, the goal of this work was to analyze these actin and tubulin genes, their expression at the mRNA and protein levels, and their parasite localization in intercellular interaction and cytokinesis. Representative bonafide actin (tvact1) and tubulin (tvtubα1) genes were cloned into and expressed in Escherichia coli. The recombinant proteins TvACT1r and TvTUBα1r were affinity purified and used as antigens to produce polyclonal antibodies. These antibodies were used in 1DE and 2DE WB and indirect immunofluorescence assays (IFA). By IFA, actin was detected as a ring on the periphery of ameboid, ovoid, and cold-induced cyst-like parasites, on pseudopods of amoeboid parasites, and in cytoplasmic extensions (filopodia) in cell–cell interactions. Tubulin was detected in the axostyle, flagellum, undulating membrane, and paradesmose during mitosis. Paradesmose was observed by IFA mainly during cytokinesis. By scanning electron microscopy, a tubulin-containing nanotubular structure similar to the tunneling nanotubes (TNTs) was also detected in the last stage of cytokinesis. In conclusion, actin and tubulin are multigene families differentially expressed that play important roles in intercellular interactions and cytokinesis.

Actin and tubulin proteins from

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from Mexico. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T. cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

Autophagy is an adaptive response for cell survival in which cytoplasmic components and organelles are degraded in bulk under normal and stress conditions. Trichomonas vaginalis is a parasite highly adaptable to stress conditions such as iron (IR) and glucose restriction (GR). Autophagy can be traced by detecting a key autophagy protein (Atg8) anchored to the autophagosome membrane by a lipid moiety. Our goal was to perform a morphological and cellular study of autophagy in T. vaginalis under GR, IR, and Rapamycin (Rapa) treatment using TvAtg8 as a putative autophagy marker. We cloned tvatg8a and tvatg8b and expressed and purified rTvAtg8a and rTvAtg8b to produce specific polyclonal antibodies. Autophagy vesicles were detected by indirect immunofluorescence assays and confirmed by ultrastructural analysis. The biogenesis of autophagosomes was detected, showing intact cytosolic cargo. TvAtg8 was detected as puncta signal with the anti-rTvAtg8b antibody that recognized soluble and lipid-associated TvAtg8b by Western blot assays in lysates from stress-inducing conditions. The TvAtg8b signal co-localized with the CytoID and lysotracker labeling (autolysosomes) that accumulated after E-64d treatment in GR parasites. Our data suggest that autophagy induced by starvation in T. vaginalis results in the formation of autophagosomes for which TvAtg8b could be a putative autophagy marker.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi is one of the most important neglected parasitic diseases in the Americas. Vaccines represent an attractive complementary or even an alternative for the control of T. cruzi infection and pre-clinical studies in mice demonstrated that trypomastigote surface antigen (TSA-1) and the flagellar calcium-binding (Tc24) parasite antigens are promising candidates for vaccine development. We performed here the first evaluation of the safety and immunogenicity of two recombinant vaccine antigens (named TSA1-C4 and Tc24-C4) in naïve non-human primates. Three rhesus macaques received 3 doses of each recombinant protein, formulated with E6020 (Eisai Co., Ltd.), a novel Toll-like receptor-4 agonist, in a stable emulsion. All parameters from blood chemistry and blood cell counts were stable over the course of the study and unaffected by the vaccine. A specific IgG response against both antigens was detectable after the first vaccine dose, and increased with the second dose. After three vaccine doses, stimulation of PBMCs with a peptide pool derived from TSA1-C4 resulted in the induction of TSA1-C4-specific TNFα-, IL-2-and IFNγ-producing CD4(+) in one or two animals while stimulation with a peptide pool derived from Tc24-C4 only activated IFNγ-producing CD4(+)T cells in one animal. In two animals there was also activation of TSA1-C4-specific IL2-producing CD8(+) T cells. This is the first report of the immunogenicity of T. cruzi-derived recombinant antigens formulated as an emulsion with a TLR4 agonist in a non-human primate model. Our results strongly support the need for further evaluation of the preventive efficacy of this type of vaccine in non-human primates and explore the effect of the vaccine in a therapeutic model of naturally-infected Chagasic non-human primates, which would strengthen the rationale for the clinical development as a human vaccine against Chagas disease.

Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.

Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

Triosephosphate isomerase of Trichomonas vaginalis ( Tv TIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2 , that participates in glucose metabolism. Tv TIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated Tv TIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of Tv TIM in T. vaginalis . Affinity ligand assays demonstrated that the recombinant Tv TIM1 and Tv TIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti- Tv TIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins ( Tv TIM1r and Tv TIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that Tv TIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis .

The recombinant ppTvCP4 (ppTvCP4r) protein, a specific inhibitor of the proteolytic activity and virulence properties of Trichomonas vaginalis, depending on cathepsin L-like cysteine proteinases (CPs) (http:dx.doi.org/ 10.1016/j.biocel.2014.12.001 [1], http:dx.doi.org/ 10.1016/j.micinf.2013.09.002 [2], http:dx.doi.org/ 10.1155/2015/946787 [3]) was stable in the elution buffer up to two months at 4 °C. However, it was prone to aggregate in PBS (functional assay buffer) [1]. Therefore, before functional assays, the aggregation kinetic of refolded ppTvCP4r was determined after the exchange to PBS. Samples of purified and refolded ppTvCP4r (0.15 mg/ml) in PBS were incubated for 0–24 h at 4 and 25 °C, spun down, measured the protein concentration in the supernatant and checked for the presence of aggregated protein in the pellet. The concentration of protein progressively decreased in the supernatant through time at both temperatures as the protein aggregated. Data in this article are related to the research paper [1]. Keywords: PpTvCP4r, Buffer exchange, Protein aggregation, Trichomonas vaginalis

Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions.

Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5&alpha, pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 &deg, C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

The release kinetics of pDNA in a bead mill was studied. Samples taken during the process were analyzed to determine total pDNA (pDNA(t)) and supercoiled pDNA (pDNA(sc)) concentration. In order to identify important variables of the process and to develop an empirical model for optimal pDNA(t) and pDNA(sc) release, a two level 23 factorial design was used with variables: mill frequency, cell concentration, and bead size. The results were analyzed by response surface methodology. The optimized conditions for pDNA(t) yield 13.26 mg/g dcw (93.41% recovery), with a mill frequency of 30 Hz, a bead size of 0.10–0.25 mm, and a cell concentration of 20 g wcw/L. However, the optimized conditions for pDNA(sc) yield 7.65 mg/g dcw (92.05% recovery), with a mill frequency of 15 Hz, a bead size of 0.10–0.25 mm, and a cell concentration of 10 g wcw/L. Cell disruption in a bead mill was proved efficient for the release of pDNA(t) and pDNA(sc) compared to the alkaline treatment. The results obtained suggest a compromise between pDNA(sc) purity and recuperation in the process development.

The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens.

Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97, 65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram. Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.

We focus on the iron response ofTrichomonas vaginalisto gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

Trypanosoma cruzi antigens TSA-1 and Tc24 have shown promise as vaccine candidates in animal studies. We evaluated here the recall immune response these antigens induce in Chagasic patients, as a first step to test their immunogenicity in humans. We evaluated the in vitro cellular immune response after stimulation with recombinant TSA-1 (rTSA-1) or recombinant Tc24 (rTc24) in mononuclear cells of asymptomatic Chagasic chronic patients (n = 20) compared to healthy volunteers (n = 19) from Yucatan, Mexico. Proliferation assays, intracellular cytokine staining, cytometric bead arrays, and memory T cell immunophenotyping were performed by flow cytometry. Peripheral blood mononuclear cells (PBMC) from Chagasic patients showed significant proliferation after stimulation with rTc24 and presented a phenotype of T effector memory cells (CD45RA-CCR7-). These cells also produced IFN-γ and, to a lesser extent IL10, after stimulation with rTSA-1 and rTc24 proteins. Overall, both antigens recalled a broad immune response in some Chagasic patients, confirming that their immune system had been primed against these antigens during natural infection. Analysis of HLA-A and HLA-B allele diversity by PCR-sequencing indicated that HLA-A03 and HLA-B07 were the most frequent supertypes in this Mexican population. Also, there was a significant difference in the frequency of HLA-A01 and HLA-A02 supertypes between Chagasic patients and controls, while the other alleles were evenly distributed. Some aspects of the immune response, such as antigen-induced IFN-γ production by CD4+ and CD8+ T cells and CD8+ proliferation, showed significant association with specific HLA-A supertypes, depending on the antigen considered. In conclusion, our results confirm the ability of both TSA-1 and Tc24 recombinant proteins to recall an immune response induced by the native antigens during natural infection in at least some patients. Our data support the further development of these antigens as therapeutic vaccine against Chagas disease., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected tropical disease, affecting mostly marginated populations and there are no effective drugs or vaccines for prevention and/or treatment. Chronic Chagasic cardiomyopathy is the mortal form of the disease; it affects 30% of infected patients and develops after 20–30 years of infection. We propose the development of a therapeutic vaccine to prevent disease progression. Our vaccine is based on the combination of two parasite proteins: Tc24 and TSA-1. In this study we assessed the response of mononuclear cells from Mexican Chagasic patients to the stimulation by both antigens. We found that both proteins are able to recall a secondary immune response, induced during natural infection in some patients, which is characterized by memory cells that can proliferate, differentiate and produce cytokines. We also showed that the immune response is associated with some of the histocompatibility allele supertypes specific of our study population. Our results support the further development and testing of both vaccine candidate antigens in future clinical trials.

The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like CPs from Trichomonas vaginalis (Cárdenas-Guerra et al., 2015 [1]). Here, we present the dataset of the trichomonad ppTvCP4r inhibitory effect against the CP proteolytic activities from other microorganisms, such as Naegleria fowleri and Acanthamoeba castellanii free-living amoeba. The proteolytic activity inhibition of total crude extracts (TCEs) of N. fowleri and A. castellanii was determined and recorded using a fluorogenic substrate specific for cathepsin L CPs without or with a ppTvCP4r treatment at different concentrations and pH.

Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis.

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 39 kDa 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 10 kDa 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.

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli , the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9 β1 ) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9 α + β1 ). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9 β2 ) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9 α + β2 ) suggesting that enzymatic activity is gained as slow transformation occurs.

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α-helix 1 against α-helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a “ball and socket” interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein. Proteins 2014; 82:22–33. © 2013 Wiley Periodicals, Inc.

American trypanosomiasis, or Chagas disease, is caused by Trypanosoma cruzi, and a vaccine would greatly improve disease control. While some studies in mice suggest that a vaccine is feasible, limited efficacy has been observed in dogs. We evaluated here the safety and efficacy of a DNA vaccine encoding TSA-1 and Tc24 antigens in a dog model of acute T. cruzi infection. Mongrel dogs were immunized with two doses of 500 μg of DNA vaccine, two weeks apart, and infected with T. cruzi (SylvioX10/4 strain) two weeks after the second vaccine dose. Another group of dogs was infected first and treated with the vaccine. Disease progression was monitored for up to 70 days post-infection. The vaccine did not induce any critical change in blood parameters, nor exacerbation of disease in vaccinated animals. On the contrary, it prevented anemia and a decrease in lymphocyte counts following T. cruzi infection in vaccinated dogs. Both preventive and therapeutic vaccination significantly reduced parasitemia, cardiac inflammation and cardiac parasite burden, and tended to reduce the development of cardiac arrhythmias. These results indicate that a preventive or therapeutic DNA vaccine encoding TSA-1 and Tc24 antigens is safe and may reduce both parasite transmission and the clinical progression of Chagas disease in vaccinated dogs. This DNA vaccine may thus be an excellent veterinary vaccine candidate. These data also further strengthen the feasibility of a Chagas disease vaccine for humans.

This review focused on potential regulatory mechanisms of Trichomonas vaginalis virulence properties, cytoadherence, cytotoxicity, phagocytosis, hemolysis, induction of apoptosis, and immune evasion in response to environmental factors of the human urogenital tract, iron, zinc, and polyamines. Understanding the multifactorial nature of trichomonal pathogenesis and its regulation may help to unravel the survival strategies of trichomonads and to implement prevention policies, opportune diagnosis, and alternative treatments for control of trichomoniasis.

An economic, simple, quantitative, and non-chromatographic method for the determination of alcohols using microdiffusion principle has been adapted and validated for acetone-butanol-ethanol (ABE) fermentation samples. This method, based on alcohols oxidation using potassium dichromate in acid medium, and detection by spectrophotometry, was evaluated varying, both, temperature (35°C, 45°C, and 55°C) and reaction time (0 to 125min). With a sample analysis time of 90min at 45°C, a limit of detection (LOD), and a limit of quantification (LOQ) of 0.10, and 0.40g/L, respectively. The proposed method has been successfully applied to determine butanol and ethanol concentrations in ABE fermentation samples with the advantage that multiple samples can be analyzed simultaneously. The measurements obtained with the proposed method were in good agreement with those obtained with the Gas Chromatography Method (GCM). This proposed method is useful for routine analysis of alcohols and screening samples in laboratories and industries.

Plasmid DNA (pDNA) vaccines are promising means to prevent and treat infectious diseases, such as leishmaniasis, but immunisation protocols require large amounts of supercoiled plasmid DNA (scpDNA). Although pDNA can be produced at a reasonable cost in bioreactors; this scale of production may not be the best method at the initial step of a vaccine development when many antigens need to be tested. Then, with the goal of improving the production of VR1012-NH36 and pVAX-NH36 pDNA vaccines against leishmaniasis, the effect of the culture medium and temperature on the pDNA yield was studied in flask cultures. The results indicate that the plasmid volumetric yield increased up to 65 mg/l in flask cultures by using a semi-defined medium, and shifting the culture temperature from 37 to 42°C at the late exponential growth phase. This pDNA production, with at least 80% of sc-pDNA at a laboratory scale seems sufficient to evaluate this and other pDNA vaccine candidates in the initial steps of vaccine development.Keywords: Plasmid DNA vaccine, plasmid DNA production, growth medium, flask culture, trace metal optimisation, leishmaniasisAfrican Journal of Biotechnology Vol. 12(31), pp. 4874-4880

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBDCex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r2 > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k N) and aggregation (k A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.

The Escherichia coli expression system remains the first choice for the production of recombinant proteins when biological activity is not compromised by posttranslational modification. Many strains of E. coli, vectors and culture conditions are available to express recombinant proteins in a soluble and correctly folded conformation. Often these strategies fail, and misfolded recombinant proteins aggregate into inclusion bodies. To recover its biological activity, a recombinant protein must be refolded, a step that has become the major limitation of the E. coli expression system. Chromatographic refolding, assisted by immobilized chaperones and foldases, is an in vitro refolding protocol that significantly improves refolding yields, yet its application at the bioprocess scale has been limited. Therefore, new cost-efficient alternatives to utilize chromatographic refolding assisted by chaperones and foldases might improve the production of biologically active proteins in E. coli. In this work, the GroEL apical domain (AD) and the oxidoreductases DsbA and DsbC fused to a carbohydrate-binding module (CBM) were purified and immobilized on microcrystalline cellulose particles. A column packed with a 60:40 (v/v) mixture of gel filtration media and cellulose particles with immobilized AD significantly improved the chromatographic refolding of rhodanese. A similar column with equimolar amounts of AD, DsbA and DsbC immobilized on cellulose particles significantly improved the oxidative chromatographic refolding of lysozyme. The assisted refolding yields were up to 80% for rhodanese and 100% for lysozyme, compared with 33% and 23%, respectively, obtained in the experiments without immobilized chaperones. In addition, AD, DsbA and DsbC immobilized on cellulose exhibited significant operational stability under the extreme denaturing conditions used in the chromatographic refolding batches. These results suggest that chromatographic refolding assisted by AD, DsbA, and DsbC immobilized on cellulose is suitable for the oxidative refolding of proteins expressed in E. coli as inclusion bodies.

The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate : ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60 degrees C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.

Introduccion: La clasificacion NICE propone unos criterios endoscopicos para predecir invasion profunda de la submucosa en los polipos del colon. Estos criterios podrian ayudar a decidir con mayor seguridad el tratamiento mas adecuado (endoscopico o quirurgico). Objetivo: Evaluar la validez diagnostica de la clasificacion NICE para predecir invasion profunda de la submucosa en polipos del colon evaluados ex vivo. Material y Metodo: Endoscopistas con una experiencia variable evaluaron la categoria NICE (1/2/3) y grado de confianza (alto/bajo) de 40 imagenes de polipos del colon. Se tomo como gold standard la histologia (9 hiperplasicos, 22 adenomas/adenocarcinomas superficiales y 9 adenocarcinomas profundos). Posteriormente realizaron un sencillo aprendizaje de la clasificacion y volvieron a valorar las mismas imagenes. Para ambos tests se registraron las respuestas contestadas con alta confianza y se calculo la S/E/VPP/VPN. Resultados: Sesenta y ocho endoscopistas cumplieron el protocolo del estudio (47% con experiencia en NBI y 19% en la clasificacion NICE). Los resultados se resumen en la Tabla 1. Conclusiones: La clasificacion NICE es util para predecir invasion profunda de la submucosa en lesiones evaluadas ex vivo. Futuros estudios deben evaluar su validez en lesiones in vivo.

SUMMARYRNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 inTrypanosoma brucei(TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics.In silicoanalysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of fiveα-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms ofT. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity.

The Zymomonas mobilis gene inv B, encoding an extracellular invertase, was translationally fused to the cellulose binding domain Cex (CBD Cex ) from Cellulomonas fimi and expressed in Escherichia coli . The fusion protein obtained, termed INVB-CBD, was purified and immobilized to Avicel (crystalline cellulose) in a single step. INVB-CBD showed an invertase activity of 20.8 IU/mL and was able to bind Avicel at a ratio of 5.3 mg of protein per gram of Avicel in 5 min. Both free and immobilized INVB-CBD showed the same optimal pH (5.5) than native INVB. However, an optimal temperature shift of −10 °C was observed for both free and immobilized INVB-CBD (45 °C) compared with the native INVB (55 °C). The K m value for the free and immobilized INVB-CBD was 193 and 81 mM of sucrose, respectively; whereas, the K m value for the native INVB was 208 mM of sucrose. Thus, suggesting an improvement in the substrate affinity of INVB after the addition of the CBD Cex tag. Additionally, a decrease in the V m value was found for the free and immobilized INVB-CBD compared with the native INVB.

The extracellular invertase from Zymomonas mobilis (INVB) was used as a model protein to study the recombinant protein production by Escherichia coli. The effect of temperature, induction and post-induction time on expression of soluble INVB protein was studied in a bench-top fermenter. The maximum productivity of soluble protein (2.7 g l−1 h−1) was obtained by decreasing the temperature (0.05 °C min−1) from 37 to 27 °C after induction with IPTG at the beginning of stationary phase. Under these conditions, the productivity of soluble INVB protein was increased by factor of 10, in relation to the process carried out overall at 37 °C.

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD(Cex), of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.

Summary Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 µM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.

The extraction and purification of phycocyanin from Calothrix sp., cyanobacteria isolated from rice fields in Cuernavaca, Morelos, Mexico is described. Phycocyanin was extracted with 2 mg of lysozyme/g wet biomass, and purified by anion chromatography using Q-Sepharose fast-flow (Pharmacia ® , 1.5 cm ×10 cm) column and hydrophobic interaction chromatography with methyl macro-prep (Bio-Rad ® , 1.5 cm ×20 cm) column. The purified protein showed a p I of 5.2 and has two subunits with apparent molecular mass of 21–17 kDa each. The estimated molecular mass of native purified phycocyanin was 114 kDa, suggesting a stereochemistry of (αβ) 3 .