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Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

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A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

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The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bß3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bß3 preventing the interaction between α2bß3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.

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The use of various herbs and their compounds has been a strategy widely used in the fight against various human diseases. For example, rosmarinic acid, a bioactive phenolic compound commonly found in Rosemary plants (Rosmarinus officinalis Labiatae), has multiple therapeutic benefits in different diseases, such as cancer. Therefore, the study aimed to evaluate in silico and in vitro the inhibition potential of the enzyme Elastase from the porcine pancreas by rosmarinic acid isolated from the plant species R. officinalis Linn. Through Molecular Docking, the mechanism of action was investigated. In addition, rosmarinic acid presented a range of 5-60 µg/mL and significantly inhibited Elastase. At 60 µg/mL, there was an inhibition of 55% on the enzymatic activity. The results demonstrate the inhibition of Elastase by rosmarinic acid, which can lead to the development of new enzyme inhibitors that can be an inspiration for developing various drugs, including anticancer drugs.

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BACKGROUND Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

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Degrons are short peptide sequences that signalize target sites for protein degradation by proteases. Herein, we bring forth the discussion on degrons present in proteins related to the immune system of Mus musculus that are potential targets for cysteine and serine proteases of Leishmania spp. and their possible roles on host immune regulation by parasites. The Merops database was used to identify protease substrates and proteases sequence motifs, while MAST/MEME Suite was applied to find degron motifs in murine cytokines (IFN-y, IL-4, IL-5, IL-13, IL-17) and transcription factors (NF-kappaB, STAT-1, AP-1, CREB, and BACH2). STRING tool was used to construct an interaction network for the immune factors and SWISS-MODEL server to generate three-dimensional models of proteins. In silico assays confirm the occurrence of degrons in the selected immune response factors. Further analyses were conducted only in those with resolved three-dimensional structures. The predicted interaction network of degron-containing M. musculus proteins shows the possibility that the specific activity of parasite proteases could interfere with the trend of Th1/Th2 immune responses. Data suggest that degrons may play a role in the immune responses in leishmaniases as targets for parasite proteases activity, directing the degradation of specific immune-related factors.

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Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

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Studies have previously demonstrated the importance of serine proteases in Leishmania. A well-known serine protease inhibitor, TPCK, was used in the present study to evaluate its in vitro and in vivo antileishmanial effects and determine its mechanism of action. Despite slight toxicity against mammalian cells (CC50 = 138.8 µM), TPCK was selective for the parasite due to significant activity against L. amazonensis and L. infantum promastigote forms (IC50 = 14.6 and 31.7 µM for L. amazonensis PH8 and Josefa strains, respectively, and 11.3 µM for L. infantum) and intracellular amastigotes (IC50 values = 14.2 and 16.6 µM for PH8 and Josefa strains, respectively, and 21.7 µM for L. infantum). Leishmania parasites treated with TPCK presented mitochondrial alterations, oxidative stress, modifications in lipid content, flagellar alterations, and cytoplasmic vacuoles, all of which are factors that could be considered as contributing to the death of the parasites. Furthermore, BALB/c mice infected with L. amazonensis and treated with TPCK had a reduction in lesion size and parasite loads in the footpad and spleen. In BALB/c mice infected with L. infantum, TPCK also caused a reduction in the parasite loads in the liver and spleen. Therefore, we highlight the antileishmanial effect of the assessed serine protease inhibitor, proposing a potential therapeutic target in Leishmania as well as a possible new alternative treatment for leishmaniasis.

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Proteases are virulence factors with a recognized impact on the Leishmania spp. life cycle. This study considers a set of analyses measuring phenotypic factors of L. (V.) braziliensis clinical isolates as promastigotes growth curves, murine peritoneal macrophages infection, inflammatory mediators production, and serine proteases gene expression (subtilisin 13: S13, subtilisin 28: S28, oligopeptidase B: OPB) assessing these isolates' fitness on in vitro conditions. Parasites had different behavior during the early growth phase from day zero to day three, and all isolates reached the stationary growth phase between days four and seven. Macrophages infection showed two tendencies, one of decreased infection rate and number of parasites per macrophage (Infection Index <1000) and another with a constant infection index (≥1400). TNF-α (≥10 pg/mL) detected in infections by 75% of isolates, IL-6 (≥80 pg/mL) by 30% of isolates and low levels of NO (≥0.01µM) in almost all infections. Gene expression showed higher values of S13 (≥2RQ) in the intracellular amastigotes of all the isolates evaluated. On the contrary, S28 expression was low (≤1RQ) in all isolates. OPB expression was different between promastigotes and intracellular amastigotes, being significantly higher (≥2RQ) in the latter form of 58% of the isolates. Predictive structural assays of S13 and OPB were performed to explore temperature influence on gene expression and the encoded proteases. Gene expression data is discussed based on in silico predictions of regulatory regions that show plasticity in the linearity index of secondary structures of S13 and OPB 3'-untranslated regions of mRNA, dependent on temperature changes. While hairpin structures suggest an active region of mRNA for both genes above 26°C, pseudoknot structure found in S13 is an indication of a particular profile of this gene at mammalian host temperatures (37°C). Furthermore, the predicted 3D structures are in accordance with the influence of these temperatures on the catalytic site stability of both enzymes, favoring their action over peptide substrates. Data gathered here suggest that L. (V.) braziliensis serine proteases can be influenced by the temperature conditions affecting parasite fitness throughout its life cycle.

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Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.