Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca²⁺-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn²⁺/Ni²⁺. The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication. Chagas disease is a neglected tropical parasitic infection. An estimated 10 million people are infected worldwide, and more than 25 million people are at risk of acquiring the disease. The therapeutic agents used to treat the disease may not be effective in all cases and also produce considerable side effects. Therefore, it is important to identify the key factors in the life cycle of the parasite that could be targets for new chemotherapeutic strategies. This paper provides evidence that a new cytoplasmic catalytic subunit of T. cruzi calcineurin (TcCaNA2) may play a critical role in host cell invasion by metacyclic trypomastigotes. Metacyclic forms with reduced TcCaNA2 expression following treatment with antisense morpholino oligonucleotides had significantly decreased capacity to invade HeLa cells. Epimastigote proliferation was inhibited to some extent by treatment with an antisense morpholino oligonucleotide targeted to TcCaNA2, but to a lesser degree than by calcineurin inhibitors (CsA, FK506 and INCA-6). The structural differences between TcCaNA2 and its human ortholog CaNA were analyzed to determine the potential of this newly identified calcineurin subunit as a chemotherapeutic target.

The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of Leishmania amazonensis, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC₅₀) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite’s morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca²⁺-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

Chagas disease caused by the obligate intracellular flagellate protozoan Trypanozoma cruzi infects about 6 million people. From the 1930s to the present, the antitumor capacity of T. cruzi has been studied; however, the identification of the responsible molecules for this effect remains undiscovered. Calcineurin, a calcium/calmodulin‐dependent serine/threonine phosphatase, is a heterodimer consisting of a catalytic subunit (CaNA) and a regulatory subunit (CaNB). It has been described that T. cruzi CaN is involved in the cell invasion and proliferation of the parasite. Recently, extracellular human CaNB has been demonstrated to be capable of inhibiting tumor growth cells, conferring an antitumor effect; however, the extracellular role of T. cruzi CaNB (TcCaNB) is still unknown. The objective of this work was to investigate the antitumor potential of TcCaNB by interacting with membrane proteins and evaluating its effects on the viability, proliferation, and morphology of tumor cells in vitro. Additionally, the possible mechanism of action of TcCaNB was explored. Murine melanoma (B16‐F10), human cervical adenocarcinoma (HeLa), and African green monkey kidney epithelial (Vero) cell lines were employed for in vitro assays. Far Western blot and immunofluorescence were performed to assess the interaction of TcCaNB with membrane proteins, and the effect of TcCaNB on cell viability and proliferation was evaluated using the MTS assay and the CyQUANT NF assay, respectively. The effect of the caspase inhibitor Z‐VAD‐FMK on TcCaNB‐stimulated tumor cells was investigated to determine if TcCaNB‐induced cell death was associated with apoptosis. To assess cell cycle progression, TcCaNB‐treated cells were analyzed by flow cytometry. In this study, the results showed an interaction of TcCaNB with cell membrane proteins in B16‐F10 and HeLa tumor lines, indicating that TcCaNB is capable of decreasing viability and proliferation of B16‐F10 and HeLa cells, with no significant effect observed in Vero cells. Furthermore, morphological changes were observed in tumor cells treated with TcCaNB. DNA fragmentations and inhibition of caspases with Z‐VAD‐FMK partially counteracted the cytotoxic effects of TcCaNB on tumor cells, suggesting that TcCaNB‐induced cell death might be associated with apoptosis. Additionally, TcCaNB caused S phase cell cycle arrest in HeLa cells, with an increase in the sub‐G1 population indicative of apoptosis, while no significant effects were observed in Vero cells.