Assessing nystatin cream treatment efficacy against Leishmania (L.) amazonensis infection in BALB/c model.

The current scenario for cutaneous leishmaniasis treatment includes the use of first and second-choice drugs, both therapeutic strategies presenting several adverse effects and being related to an increment of treatment-refractory parasite strains. These facts encourage the search for new treatment approaches, including repositioning drugs, such as nystatin. Although in vitro assays show that this polyene macrolide compound has leishmanicidal activity, no in vivo evidence for a similar activity has been shown so far for the commercial nystatin cream formulation. This work assessed the effects of nystatin cream (25,000 IU/g) administered on mice in an amount to completely cover the paw surface of BALB/c mice infected with Leishmania (L.) amazonensis once a day, until a total of up to 20 doses. The data presented herein points to unequivocal evidence that treatment with this formulation causes a statistically significant reduction of swelling/edema in mice paws when compared to animal groups not submitted to this treatment regimen after the fourth week of infection: lesion sizes at the sixth (p = 0.0159), seventh (p = 0.0079) and eighth (p = 0.0079) week. Furthermore, swelling/edema reduction relates to a decrease in parasite load in the footpad (∼48%) and in draining lymph nodes (∼68%) at eight weeks post-infection. This is the first report of the effectiveness of nystatin cream used as a topical treatment in BALB/c model for cutaneous leishmaniasis.

Detection of Antibodies against Hepatitis A Virus (HAV) by a Surface Plasmon Resonance (SPR) Biosensor: A New Diagnosis Tool Based on the Major HAV Capsid Protein VP1 (SPR-HAVP1).

Hepatitis A (HA) is an acute human infectious disease caused by a positive single-stranded RNA virus (HAV). It is mainly acquired through the fecal-oral route and is primarily spread by contact between people and exposure to contaminated water and food. Recently, large outbreaks of HA have been reported by low and moderate endemicity countries, emphasizing its importance in public health and the need for rapid and large-scale diagnostic tests to support public health decisions on HA. This work proposes a new tool for HAV diagnosis based on the association of surface plasmonic resonance with major capsid protein VP1 (SPR-HAVP1 assay), detecting IgM antibodies for HAV in human serum samples. Structural analyses of VP1 B-lymphocyte epitopes showed continuous and discontinuous epitopes. The discontinuous epitopes were identified in the N-terminal region of the VP1 protein. Both epitope types in the VP1 protein were shown by the reactivity of VP1 in native and denaturing conditions to IgM anti-HAV, which was favorable to tests of VP1 in the SPR assays. SPR-HAVP1 assays showed good performance in the detection of IgM polyclonal antibody anti-HAV. These assays were performed using a COOH5 sensor chip functionalized with VP1 protein. The sensorgram record showed a significant difference between positive and negative serum samples, which was confirmed by analysis of variation of initial and final dissociation values through time (ΔRUd/t). The data gathered here are unequivocal evidence that the SPR-HAVP1 strategy can be applied to detect IgM antibodies in human serum positive to the HAV. This is a new tool to be explored to diagnose human HAV infections.

SARS-CoV-2 Proteins Bind to Hemoglobin and Its Metabolites.

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

In Silico Insights into the Mechanism of Action of Epoxy-α-Lapachone and Epoxymethyl-Lawsone in Leishmania spp.

Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have been shown to be promising drugs for Leishmaniases treatment. Although, it is known the action spectrum of both compounds affect the Leishmania spp. multiplication, there are gaps in the molecular binding details of target enzymes related to the parasite's physiology. Molecular docking assays simulations were performed using DockThor server to predict the preferred orientation of both compounds to form stable complexes with key enzymes of metabolic pathway, electron transport chain, and lipids metabolism of Leishmania spp. This study showed the hit rates of both compounds interacting with lanosterol C-14 demethylase (-8.4 kcal/mol to -7.4 kcal/mol), cytochrome c (-10.2 kcal/mol to -8.8 kcal/mol), and glyceraldehyde-3-phosphate dehydrogenase (-8.5 kcal/mol to -7.5 kcal/mol) according to Leishmania spp. and assessed compounds. The set of molecular evidence reinforces the potential of both compounds as multi-target drugs for interrupt the network interactions between parasite enzymes, which can lead to a better efficacy of drugs for the treatment of leishmaniases.

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression.

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

Antileishmanial Activity of 2-Methoxy-4H-spiro-[naphthalene-1,2'-oxiran]-4-one (Epoxymethoxy-lawsone): A Promising New Drug Candidate for Leishmaniasis Treatment.

Epoxymethoxylawsone is a naphthoquinone derivative promising as drug candidate for the treatment of leishmaniases. In the present work the effectiveness of epoxymethoxylawsone, and meglumine antimoniate on Leishmania (Leishmania) amazonensis parasites and on mice paw lesions of infected BALB/c mice was assessed. In an intracellular amastigotes assay, the half-maximal inhibitory concentration (IC50) value for epoxymethoxylawsone was slightly higher (1.7-fold) than that found for meglumine antimoniate. The efficacy of both drugs became more evident after 48 h of exposure when either the oxirane compound and reference drug reached 18-fold and 7.4-fold lower IC50 values (0.40 ± 0.001 µM and 0.60 ± 0.02 µM), respectively. Promastigotes were also affected by epoxymethoxylawsone after 24 h of incubation (IC50 = 45.45 ± 5.0 µM), but with IC50 6-fold higher than those found for intracellular amastigotes. Cytotoxicity analysis revealed that epoxymethoxylawsone (CC50 = 40.05 ± µM) has 1.7-fold higher effects than meglumine antimoniate (CC50 = 24.14 ± 2.6 µM). Treatment of the paw lesion in infected BALB/c mice with epoxymethoxy-lawsone led to a significant 27% reduction (p < 0.05) of the lesion size, for all administrated doses, compared to the control group. Lesion reduction was also detected after mice treatment with meglumine antimoniate, reaching 31.0% (0.23 mg of Sb(V)/Kg/day and 2.27 mg of Sb(V)/Kg/day) and 64.0% (22.7 mg of Sb(V)/Kg/day). In addition, mice lesion ultrastructural changes were evidenced in amastigotes. The set of data gathered here indicate that epoxymethoxylawsone has pronounced effects on parasites and merits furthering to the preclinical stage.

Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-ß knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

Epoxy-α-lapachone has in vitro and in vivo anti-leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm(2)), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm(2)) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm(2)). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection.

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D(b):Ig and H2 L(d):Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis.

Evidences for leishmanicidal activity of the naphthoquinone derivative epoxy-α-lapachone.

In this work, we analyze the leishmanicidal effects of epoxy-α-lapachone on Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis. Promasigotes and amastigotes (inhabiting human macrophages) from both species were assayed to verify the compound's activity over the distinct morphological stages. The incubation with epoxy-α-lapachone led to a significant decrease in the numbers of promastigotes from both species in the cultures, in a dose-and time-dependent fashion. The survival of amastigotes inhabiting human macrophages was also drastically affected by the compound, as shown by the variations in the endocytic index. Our results indicate that the epoxy-α-lapachone has an antiparasitic effect over Leishmania in both morphological stages and may potentially affect a range of species in two distinct subgenera of this parasite.

Efficacy of the treatment using a microemulsion loaded with epoxy-α-lapachone in combination with meglumine antimoniate against murine infection by Leishmania (Leishmania) amazonensis.

Leishmaniasis is a disease caused by Leishmania spp., affecting millions of people around the world. For decades, its treatment has been based on pentavalent antimonials, which notoriously cause toxic side effects in patients. In this study, epoxy-α-lapachone incorporated into an oil-in-water-type microemulsion (ELAP-ME) and meglumine antimoniate (MA) were assayed in monotherapy and in combination (ELAP-ME/MA) in BALB/c mice infected with Leishmania (Leishmania) amazonensis. In general, there was a reduction in paw lesion size (up to 37% reduction) and decreases of parasite loads in the footpad (∼40%) and lymph nodes (∼31%) of animals treated with ELAP-ME/MA, when compared to the non-treated control groups. Analyses of serum biochemical parameters revealed that the ELAP-ME/MA showed lower renal and hepatic toxicity when compared to MA 2-doses/week monotherapy. These findings indicate that the ELAP-ME/MA combination may be a promising approach for the treatment of cutaneous leishmaniasis.

Epoxy-α-lapachone (2,2-Dimethyl-3,4-dihydro-spiro[2H-naphtho[2,3-b]pyran-10,2'-oxirane]-5(10H)-one): a promising molecule to control infections caused by protozoan parasites.

Natural products and their derivatives have been sources of search and research for new drugs for the treatment of neglected diseases. Naphthoquinones, a special group of quinones, are products of natural metabolites with a wide spectrum of biological activities and represent a group of interesting molecules for new therapeutic propositions. Among these compounds, lapachol stands out as a molecule from the heartwood of Tabebuia sp. whose structural changes resulted in compounds considered promising, such as epoxy-α-lapachone (ELAP). The biological activity of ELAP has been demonstrated, so far, for parasitic protozoa such as Leishmania spp., Trypanosoma cruzi and Plasmodium spp., species causing diseases needing new drug development and adequate health policy. This work gathers in vitro and in vivo studies on these parasites, as well as the toxicity profile, and the probable mechanisms of action elucidated until then. The potential of ELAP-based technology alternatives for a further drug is discussed here.

Degron Pathways and Leishmaniasis: Debating Potential Roles of Leishmania spp. Proteases Activity on Guiding Hosts Immune Response and Their Relevance to the Development of Vaccines.

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.

Development of a microemulsion loaded with epoxy-α-lapachone against Leishmania (Leishmania) amazonensis murine infection.

Epoxy-α-lapachone (ELAP), an oxirane-functionalized molecule synthesized from naturally occurring lapachol, has shown promising activity against murine infection with Leishmania (Leishmania) amazonensis. Herein, we report the successful development of oil-in-water-type (o/w) microemulsions (ME) loaded with ELAP (ELAP-ME) using Capmul MCM, Labrasol, and PEG 400. Stability studies revealed that ELAP-ME (100 µg/mL of ELAP), which was comprised of globule size smaller than 120.4 ± 7.7 nm, displayed a good stability profile over 73 days. ELAP-ME had an effect in BALB/c mice infected with L. (L.) amazonensis, causing reductions in paw lesions after two weeks of treatment (∼2-fold) when compared to untreated animals. Furthermore, there was also a reduction in the parasite load both in the footpad (60.3%) and in the lymph nodes (31.5%). Based on these findings, ELAP-ME emerges as a promising treatment for tegumentar leishmaniasis.

Novel Insights Into Leishmania (Viannia) braziliensis In Vitro Fitness Guided by Temperature Changes Along With Its Subtilisins and Oligopeptidase B.

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.

Combining Well-Tempered Metadynamics Simulation and SPR Assays to Characterize the Binding Mechanism of the Universal T-Lymphocyte Tetanus Toxin Epitope TT830-843.

Peptide TT830-843 from the tetanus toxin is a universal T-cell epitope. It helps in vaccination and induces T-cell activation. However, the fine molecular interaction between this antigen and the major histocompatibility complex (MHC) remains unknown. Molecular analysis of its interaction with murine MHC (H-2) was proposed to explore its immune response efficiency. Molecular dynamics simulations are important mechanisms for understanding the basis of protein-ligand interactions, and metadynamics is a useful technique for enhancing sampling in molecular dynamics. SPR (surface plasmon resonance) assays were used to validate whether the metadynamics results are in accordance with the experimental results. The peptide TT830-843 unbinding process was simulated, and the free energy surface reconstruction revealed a detailed conformational landscape. The simulation described the exiting path as a stepwise mechanism between progressive detachment states. We pointed out how the terminus regions act as anchors for binding and how the detachment mechanism includes the opening of α-helices to permit the peptide's central region dissociation. The results indicated the peptide/H-2 receptor encounter occurs within a distance lesser than 27.5 Å, and the encounter can evolve to form a stable complex. SPR assays confirmed the complex peptide/H-2 as a thermodynamically stable system, exhibiting enough free energy to interact with TCR on the antigen-presenting cell surface. Therefore, combining in silico and in vitro assays provided significant evidence to support the peptide/H-2 complex formation.

Axenic amastigotes of Leishmania species as a suitable model for in vitro studies.

Leishmania spp. are etiological agents of infection diseases, which in some cases can be fatal. The main forms of their biological cycle, promastigotes and amastigotes, can be maintained in vitro. While promastigotes are easier to maintain, amastigotes are more complex and can be obtained through different ways, including infection assays of tissues or in vitro cells, and differentiation from promastigotes to axenic amastigotes. Several protocols have been proposed for in vitro differentiation for at least 12 Leishmania spp. of both subgenera, Leishmania and Viannia. In this review we propose a critical summary of axenic amastigotes induction, as well as the impact of these strategies on metabolic pathways and regulatory networks analyzed by omics approaches. The parameters used by different research groups show considerable variations in temperature, pH and induction stages, as highlighted here for Leishmania (Viannia) braziliensis. Therefore, a consensus on strategies for inducing amastigogenesis is necessary to improve accuracy and even define stage-specific biomarkers. In fact, the axenic amastigote model has contributed to elucidate several aspects of the parasite cycle, however, since it does not reproduce the intracellular environment, its use requires several precautions. In addition, we present a discussion about using axenic amastigotes for drug screening, suggesting the need of a more sensitive methodology to verify cell viability in these tests. Collectively, this review explores the advantages and limitations found in studies with axenic amastigotes, done for more than 30 years, and discuss the gaps that impair their use as a suitable model for in vitro studies.

Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony.

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders-R) and relapse or therapeutic failure (Non-responders-NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.