Induction of apoptosis by essential oil of Dracocephalum kotschyi on Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is a protist parasite that causes trichomoniasis, a sexually transmitted disease. Metronidazole is the current treatment for trichomoniasis. However, this drug can provoke severe side effects, and some strains present resistance, making the development of alternative treatments for trichomoniasis urgent. OBJECTIVES: We investigate the use of essential oil obtained from Dracocephalum kotschyi on T. vaginalis. D. kotschyi has antispasmodic and analgesic properties and is well known in Iran. METHODS: The essential oil was obtained by hydrodistillation from 1000 g of the powdered plant. Gas chromatography-mass spectrometry analysis was used for the chemical composition of the essential oil, and 11 substances were identified, corresponding to 91.5% of the oil. Copaene (22.15%), Methyl geranate (16.31%), Geranial (13.78%) and Carvone (11.34%) were the main substances. A cell viability test was used to determine the percentage of growth inhibition (GI%) and the half-maximal inhibitory concentration (IC50) on T. vaginalis after incubation with the prepared essential oil. RESULTS: The oil induced an IC50 of 84.07 µg/ml after 24 h contact with trophozoites. Cytotoxicity was determined by MTT assay on the J774.A1 haematopoietic cell line. In addition, the initial stage of apoptosis was assayed using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. Evaluation of the in vitro anti-trichomonal properties of D. kotschyi essential oils showed that it effectively induces apoptosis on T. vaginalis between 100 and 700 µg/ml after 48 h without toxicity on haematopoietic cells, suggesting that D. kotschyi essential oil can induce programmed death in T. vaginalis. CONCLUSIONS: The anti-trichomonal properties of D. kotschyi essential oil indicate that they could be suitable for new pharmacologic studies after new tests with human vaginal epithelial cells.

Giardia intestinalis and its endomembrane system.

Giardia intestinalis has unique characteristics, even in the absence of certain organelles. For instance, Golgi and mitochondria are not found. On the other hand, there is a network of peripheral vacuoles (PVs) and mitosomes. The endoplasmic reticulum (ER), nuclear membrane, peroxisomes, and lipid bodies are present. The peripheral vacuole system seems to play several simultaneous roles. It is involved in the endocytic activity of the trophozoite but also has characteristics of early and late endosomes and even lysosomes, establishing a connection with the ER. Some of the PVs contain small vesicles, acting as multivesicular bodies, including the release of exosomes. The mitosomes are surrounded by two membranes, divide during mitosis, and are distributed throughout the cell. They do not contain DNA, enzymes involved in the citric acid cycle, respiratory chain, or ATP synthesis. However, they contain the iron-sulfur complex and transporters as TOM and TIM. Some mitosomes are linked to flagellar axonemes through a fibrillar connection. During encystation, two types of larger cytoplasmic vesicles appear. One originating from the ER contains the cyst wall proteins. Another contains carbohydrates. Both migrate to the cell periphery and fuse with plasma membrane secreting their contents to give rise to the cell wall.

A review study on the anti-trichomonas activities of medicinal plants.

The parasitic diseases represent the most important health risk, especially in underdeveloped countries where they have a deep impact on public health. Trichomoniasis is a prevalent non-viral sexually transmitted disease, and a significant amount of new cases are identified each year globally. Furthermore, the infection is linked with serious concerns such as pregnancy outcomes, infertility, predisposition to cervical and prostate cancer, and increased transmission and acquisition of HIV. The therapy is restricted, adverse effects are often observed, and resistance to the drugs is emerging. Based on this, a new treatment for trichomoniasis is necessary. Natural products represent a rich source of bioactive compounds, and even today, they are used in the search for new drugs. Additionally, natural products provide a wide variety of leadership structures that can be used by the pharmaceutical industry as a template in the development of new drugs that are more effective and have fewer or no undesirable side effects compared to current treatments. This review focuses on the medicinal plants that possess anti-trichomonal activity in vitro or in vivo. An electronic database search was carried out covering the last three decades, i.e., 1990-2020. The literature search revealed that almost a dozen isolated phytoconstituents are being explored globally for their anti-trichomonal activity. Simultaneously, many countries have their own traditional or folk medicine for trichomoniasis that utilizes their native plants, as a whole, or even extracts. This review focuses mainly on the human parasite Trichomonas vaginalis. However, at some points mention is also made to Tritrichomonas foetus that causes trichomoniasis in animals of high veterinary and economical interest. We will focus on the plants and plant-based compounds and their anti-trichomonal activity. The literature search highlighted that there are abundant compounds that possess anti-trichomonal activity; however, in-depth in-vivo evaluation of compounds and their clinical evaluation has not been undertaken. There is a critical need for new anti-trichomonal compounds, and focused research on phytoconstituents can provide the way forward.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

The peripheral vesicles gather multivesicular bodies with different behavior during the Giardia intestinalis life cycle.

Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50 nm, containing intraluminal vesicles (ILVs).

G-CSF-Induced Suppressor IL-10+ Neutrophils Promote Regulatory T Cells That Inhibit Graft-Versus-Host Disease in a Long-Lasting and Specific Way.

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation, and many efforts have been made to overcome this important limitation. We showed previously that G-CSF treatment generates low-density splenic granulocytes that inhibit experimental aGVHD. In this article, we show that aGVHD protection relies on incoming IL-10+ neutrophils from G-CSF-treated donor spleen (G-Neutrophils). These G-Neutrophils have high phagocytic capacity, high peroxide production, low myeloperoxidase activity, and low cytoplasmic granule content, which accounts for their low density. Furthermore, they have low expression of MHC class II, costimulatory molecules, and low arginase1 expression. Also, they have low IFN-γ, IL-17F, IL-2, and IL-12 levels, with increased IL-10 production and NO synthase 2 expression. These features are in accordance with the modulatory capacity of G-Neutrophils on regulatory T cell (Treg) generation. In vivo, CD25+ Treg depletion shortly after transplantation with splenic cells from G-CSF-treated donors blocks suppression of aGVHD, suggesting Treg involvement in the protection induced by the G-Neutrophils. The immunocompetence and specificity of the semiallogeneic T cells, long-term after the bone marrow transplant using G-Neutrophils, were confirmed by third-party skin graft rejection; importantly, a graft-versus-leukemia assay showed that T cell activity was maintained, and all of the leukemic cells were eliminated. We conclude that G-CSF treatment generates a population of activated and suppressive G-Neutrophils that reduces aGVHD in an IL-10- and Treg-dependent manner, while maintaining immunocompetence and the graft versus leukemia effect.

Mitosomal chaperone modulation during the life cycle of the pathogenic protist Giardia intestinalis.

The mitosome is a double-membrane enveloped organelle that is found in few unicellular eukaryotes, one of which is the human intestinal parasitic protist Giardia intestinalis, which also lacks mitochondria and peroxisomes. This flagellated protist grows in vitro as trophozoites and under some conditions, differentiates into cysts, which are characterized by the absence of externalized flagella, a round shape, and the presence of a cyst wall. The presence and distribution of mitosomal proteins, such as giardial iron-sulfur cluster protein (GiIscU), heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiCpn60), during the process of trophozoite-to-cyst transformation was tracked using confocal laser scanning microscopy and western blotting. During the early stages of the differentiation process (∼12h), there was a significant decrease in the extent of chaperone labeling in the cells, which disappeared after 21h but was recovered during the cyst stage; IscU labeling remained present throughout the differentiation process. This finding was confirmed by mRNA expression analysis, thus indicating that a process modulates the expression of mitosomal chaperones during the G. intestinalis life cycle. Microscopy techniques, such as structured illumination and electron tomography, revealed a novel profile for central mitosomes, as well as the presence of both rounded and elongated mitosomes.

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvß3/VEGFR2 Axis.

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvß3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc.

Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

Changes in the structural organization of the cytoskeleton of Tritrichomonas foetus during trophozoite-pseudocyst transformation.

Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. It grows in axenic media as a trophozoite with a pear-shaped body, three anterior flagella, and one recurrent flagellum. However, under some well-controlled experimental conditions in vitro, as well as in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized but the cells do not display a cyst wall. This form, known as the endoflagellar or pseudocystic form, is viable, and can be transformed back to trophozoites with pear-shaped body. We used confocal laser scanning microscopy, and high resolution scanning electron microscopy to examine the changes that take place in the protozoan cytoskeleton during trophozoite-pseudocyst transformation. Results confirmed previous studies and added new structural information to the organization of cytoskeletal structures during the transformation process. We observed that changes take place in the pseudocysts' axostyle and costa, which acquired a curved shape. In addition, the costa of multinucleated/polymastigont pseudocysts took variable conformations while curved. The costa accessory structure, as well as a network of filaments connecting this structure to the region where the recurrent flagellum associates to the protozoan body, was not seen in pseudocysts. In addition, the axostyle was fragmented during trophozoite-pseudocyst transformation.

New insights on the Golgi complex of Tritrichomonas foetus.

Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis.

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a ∼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of ∼24 kDa with pI values of ∼6.7, 6.9, and 7.0 and two spots of ∼22 and ∼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.

The TvLEGU-1, a legumain-like cysteine proteinase, plays a key role in Trichomonas vaginalis cytoadherence.

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.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

Purpose: Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. Materials and Methods: Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. Results: For all analyzed parameters Sham and Pp groups presented no statistical difference. Conclusion: When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats. .

A new set of carbohydrate-positive vesicles in encysting Giardia lamblia.

Giardia lamblia is a protozoan parasite that presents both trophozoite and cyst forms. In this study, the distribution of the different sugar residues and the origin of the carbohydrate components of the cyst wall were studied using transmission electron microscopy, ultrastructural cytochemistry for carbohydrate detection and immunocytochemistry. Immunofluorescence microscopy using anti-cyst wall protein 1 (CWP1) and gold- and fluorescent-conjugated lectins, such as WGA and DBA, were also used. Interestingly, a population of carbohydrate-containing vesicles, distinct from the encystation-specific vesicles (ESVs) was found in the encysting cells and was named encystation carbohydrate-positive vesicles (ECVs). The differences between the ECVs and the ESVs were: (1) they are electron-translucent, whereas ESVs are electron dense; (2) they do not react with antibodies against cyst wall proteins; (3) the contents are positive for carbohydrates, whereas ESVs display a negative reaction; and (4) they exhibit a positive labeling for DBA indicating the presence of N-acetyl-galactosamine, whereas ESVs are negative. To evaluate if ECVs could be vesicles involved in the endocytic pathway, endocytic markers were used. No co-localization of these markers with ECVs was observed. We suggest that the ECVs may represent a new structure involved in cyst wall formation.

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1) protein with a unique regulatory C-terminus.

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

A protein phosphatase 1 gamma (PP1γ) of the human protozoan parasite Trichomonas vaginalis is involved in proliferation and cell attachment to the host cell.

In this work, evidence for a critical role of Trichomonas vaginalis protein phosphatase 1 gamma (TvPP1γ) in proliferation and attachment of the parasite to the mammalian cell is provided. Firstly, proliferation and attachment of T. vaginalis parasites to HeLa cells was blocked by calyculin A (CA), a potent PP1 inhibitor. Secondly, it was demonstrated that the enzyme activity of native and recombinant TvPP1γ proteins was inhibited by CA. Thirdly, reverse genetic studies confirmed that antisense oligonucleotides targeted to PP1γ but not PP1α or ß inhibited proliferation and attachment of trichomonads CA-treated parasites underwent cytoskeletal modifications, including a lack of axostyle typical labelling, suggesting that cytoskeletal phosphorylation could be regulated by a CA-sensitive phosphatase where the role of PP1γ could not be ruled out. Analysis of subcellular distribution of TvPP1γ by cell fractionation and electron microscopy demonstrated the association between TvPP1γ and the cytoskeleton. The expression of adhesins, AP120 and AP65, at the cell surface was also inhibited by CA. The concomitant inhibition of expression of adhesins and changes in the cytoskeleton in CA-treated parasites suggest a specific role for PP1γ -dependent dephosphorylation in the early stages of the host-parasite interaction. Molecular modelling of TvPP1γ showed the conservation of residues critical for maintaining proper folding into the gross structure common to PP1 proteins. Taken together, these results suggest that TvPP1γ could be considered a potential novel drug target for treatment of trichomoniasis.