The activity of a Hexameric M17 Metallo-Aminopeptidase is associated with survival of Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metalloaminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology

Advances in nanocarriers as drug delivery systems in Chagas disease

Chagas disease is one of the most important public health problems in Latin America due to its high mortality and morbidity levels. There is no effective treatment for this disease since drugs are usually toxic with low bioavailability. Serious efforts to achieve disease control and eventual eradication have been unsuccessful to date, emphasizing the need for rapid diagnosis, drug development, and a reliable vaccine. Novel systems for drug and vaccine administration based on nanocarriers represent a promising avenue for Chagas disease treatment. Nanoparticulate systems can reduce toxicity, and increase the efficacy and bioavailability of active compounds by prolonging release, and therefore improve the therapeutic index. Moreover, nanoparticles are able to interact with the host’s immune system, modulating the immune response to favour the elimination of pathogenic microorganisms. In addition, new advances in diagnostic assays, such as nanobiosensors, are beneficial in that they enable precise identification of the pathogen. In this review, we provide an overview of the strategies and nanocarrier-based delivery systems for antichagasic agents, such as liposomes, micelles, nanoemulsions, polymeric and non-polymeric nanoparticles. We address recent progress, with a particular focus on the advances of nanovaccines and nanodiagnostics, exploring new perspectives on Chagas disease treatment

Essential oils : in vitro activity against Leishmania amazonensis, cytotoxicity and chemical composition

Background: The current chemotherapy for cutaneous leishmaniosis (CL) has a series of drug limitations such as toxic side effects, long duration, high costs and drug resistance, which requires the development of new drugs or effective alternatives to the CL treatment. Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against protozoa. Thus, this study aims to evaluate the biological activity of different essential oils (EOs) on Leishmania (L.) amazonensis promastigotes forms, as well as their cytotoxicity on mammalian cells and chemical composition. Methods: Sixteen EOs were evaluated by mean of IC50/24 h and cytotoxicity against L6 cells (CC50/24 h) using Resazurin assay. Only those EOs that presented better results for IC50/24 h were submitted to GC–MS analysis to determine their chemical constitution. Results: The EO from Cinnamodendron dinisii, Matricaria chamomilla, Myroxylon peruiferum, Salvia sclarea, Bulnesia sarmientoi, Ferula galbaniflua, Siparuna guianensis and Melissa officinalis were the most active against L. amazonensis with IC50/24 h ranging from 54.05 to 162.25 μg/mL. Analysis of EOs by GC–MS showed mainly the presence of β-farnesene (52.73 %) and bisabolol oxide (12.09 %) for M. chamomilla; α-copaene (13.41 %), safrole (8.35 %) and δ-cadinene (7.08 %) for M. peruiferum; linalool (28.80 %) and linalyl acetate (60.08 %) for S. sclarea; guaiol (48.29 %) and 2-undecanone (19.49 %) for B. sarmientoi; ethyl phthalate (13.09 %) and methyl-8-pimaren-18-oate (41.82 %) for F. galbaniflua; and neral (37.18 %) and citral (5.02 %) for M. officinalis. Conclusion: The EO from F. galbaniflua showed to be effective against L. amazonensis promastigotes forms and presented low cytotoxic activity against L6 cells. Thus, it represents a strong candidate for future studies aiming its molecular activity on these pathogenic parasites

Revealing a novel Otubain-Like Enzyme from Leishmania infantum with deubiquitinating activity toward K48-linked substrate

Deubiquitinating enzymes (DUBs) play an important role in regulating a variety of eukaryotic processes. In this context, exploring the role of deubiquitination in Leishmania infantum could be a promising alternative to search new therapeutic targets for leishmaniasis. Here we present the first characterization of a DUB from L. infantum, otubain (OtuLi), and its localization within parasite. The recombinant OtuLi (rOtuLi) showed improved activity on lysine 48 (K48)-linked over K63-linked tetra-ubiquitin (Ub) and site-directed mutations on amino acids close to the catalytic site (F82) or involved in Ub interaction (L265 and F182) caused structural changes as shown by molecular dynamics, resulting in a reduction or loss of enzyme activity, respectively. Furthermore, rOtuLi stimulates lipid droplet biogenesis (an inflammatory marker) and induces IL-6 and TNF-a secretion in peritoneal macrophages, both proinflammatory cytokines. Our findings suggest that OtuLi is a cytoplasmic enzyme with K48-linked substrate specificity that could play a part in proinflammatory response in stimulated murine macrophages

Triatomines: Trypanosomatids, Bacteria, and Viruses Potential Vectors?

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatomines may be competent vectors for pathogens such as Serratia marcescens, Bartonella, and Mycobacterium leprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role infungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was over-expressed in bacteria and a polyclonal antibody to this protein was produced. Weidentified a 180-kDa protein species reactive to the antibody produced in miceagainst the P. brasiliensis recombinant purified formamidase of 45 kDa. The180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide massfinger printing using MS. The identical mass spectra generated by the 180 and the45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formami-dase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights intoformamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism

Triatomines : trypanosomatids, bacteria, and viruses potential vectors?

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatominesmay be competent vectors for pathogens such as Serratiamarcescens, Bartonella, andMycobacteriumleprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

A Deep Insight into the Sialome of Rhodnius neglectus, a vector of chagas disease

Background Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immuneresponses. Methods/Principal Findings Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva.We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. Conclusions/Significance The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts

Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening

International audienceWe have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/β-hydrolase domain and a β-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease