A synthetic peptide derived of the ß2-ß3 loop of the plant defensin from Vigna unguiculata seeds induces Leishmania amazonensis apoptosis-like cell death.

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.

Autophagic elimination of Trypanosoma cruzi in the presence of metals.

Trypanosoma cruzi is an obligate intracellular parasite transmitted to vertebrate hosts by blood-sucking insects. Molecules present in parasites and mammalian cells allow the recognition and parasite internalization. Metallic ions play an essential role in the establishment and maintenance of host-parasite interaction. However, little is known about how parasites handle with essential and nonessential metal quotas. This study aimed to investigate the influence of metal ions on the biological processes of T. cruzi infected cells. Infected cells were incubated with ZnCl2, CdCl2, and HgCl2 for 12 h and labeled with different specific dyes to investigate the cellular events related to intracellular parasite death and elimination. Infected host cells and parasite's mitochondria underwent functional and structural disorders, in addition to parasite's DNA condensation and pH decrease on host cells, which led to parasite death. Further investigations suggested that lysosomes were involved in pH decrease and the double membrane of the endoplasmic reticulum formed vacuoles surrounding damaged parasites, which indicate the occurrence of autophagy for parasite elimination. In conclusion, low concentrations of nonessential and essential metals cause a series of damage to Trypanosoma cruzi organelles, leading to its loss of viability, death, and elimination, with no removal of the host cells.

The toxic effect of Vu-Defr, a defensin from Vigna unguiculata seeds, on Leishmania amazonensis is associated with reactive oxygen species production, mitochondrial dysfunction, and plasma membrane perturbation.

Plant defensins are plant antimicrobial peptides that present diverse biological activities in vitro, including the elimination of Leishmania amazonensis. Plant defensins are considered promising candidates for the development of new drugs. This protozoan genus has great epidemiological importance and the mechanism behind the protozoan death by defensins is unknown, thus, we chose L. amazonensis for this study. The aim of the work was to analyze the possible toxic mechanisms of Vu-Defr against L. amazonensis. For analyses, the antimicrobial assay was repeated as previously described, and after 24 h, an aliquot of the culture was tested for viability, membrane perturbation, mitochondrial membrane potential, reactive oxygen species (ROS) and nitric oxide (NO) inductions. The results of these analyses indicated that after interaction with L. amazonensis, the Vu-Defr causes elimination of promastigotes from culture, membrane perturbation, mitochondrial membrane collapse, and ROS induction. Our analysis demonstrated that NO is not produced after Vu-Defr and L. amazonensis interaction. In conclusion, our work strives to help to fill the gap relating to effects caused by plant defensins on protozoan and thus better understand the mechanism of action of this peptide against L. amazonensis.

In Vitro Anti-Toxoplasma gondii and Antimicrobial Activity of Amides Derived from Cinnamic Acid.

Most cinnamic acids, their esters, amides, aldehydes, and alcohols present several therapeutic actions through anti-inflammatory, antitumor, and inhibitory activity against a great variety of microorganisms. In this work, eight amines derived from cinnamic acid were synthesized and tested against host cells infected with Toxoplasma gondii and the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and three strains of Staphylococcus aureus. Compounds 3 and 4 showed the best result against intracellular T. gondii, presenting antiparasitic activity at low concentrations (0.38 and 0.77 mM). The antibacterial activity of these compounds was also evaluated by the agar microdilution method, and amides 2 and 5 had a minimum inhibitory concentration of 250 µg mL-1 against two strains of S. aureus (ATCC 25923 and bovine strain LSA 88). These also showed synergistic action along with a variety of antibiotics, demonstrating that amines derived from cinnamic acid have potential as pharmacological agents.

Further aspects of Toxoplasma gondii elimination in the presence of metals.

Toxoplasma gondii, the etiological agent of toxoplasmosis, infects nucleated cells and then resides and multiplies within a parasitophorous vacuole. For this purpose, the parasite secretes many virulence factors for the purpose of invading and subverting the host microbicidal defenses in order to facilitate its survival in the intracellular milieu. Essential metals are structural components of proteins and enzymes or cofactors of enzymatic reactions responsible for these parasitic survival mechanisms. However, an excess of non-essential or essential metals can lead to parasite death. Thus, infected host cells were incubated with 20 µM ZnCl2 in conjunction with 3 µM CdCl2 or HgCl2 for 12 h in order to investigate cellular events and organelle damage related to intracellular parasite death and elimination. In the presence of these metals, the tachyzoites undergo lipid uptake and transport impairment, functional and structural mitochondrial disorders, DNA condensation, and acidification of the parasitophorous vacuole, thus leading to parasite death. Additional research has suggested that lysosome-vacuole fusion was involved in parasite elimination since acid phosphatases were found inside the parasitophorous vacuole, and vacuoles containing parasites were also positive for autophagy. In conclusion, low concentrations of CdCl2, HgCl2, and ZnCl2 can cause damage to Toxoplasma gondii organelles, leading to loss of viability, organelle death, and elimination without causing toxic effects to host cells.

Life and death of Trypanosoma cruzi in presence of metals.

Trypanosoma cruzi has many molecules that need metallic elements to work, allowing cell invasion and the establishment of infection, causing Chagas disease. Nonetheless, knowledge regarding how the parasites address metals and maintain homeostasis is lacking. To study this relationship, zinc, cadmium and mercury were chosen. Epimastigote, trypomastigote and intracellular forms of T. cruzi were incubated with these metals for different times and at different concentrations. In general, epimastigotes were the most sensitive and trypomastigotes the most resistant to metals. ZnCl2 induced low toxic effects to all parasite forms. Although the parasites were very sensitive to the toxic effects of CdCl2 and HgCl2, pretreatment with ZnCl2 decreased the death rate. The trypomastigotes pretreated with CdCl2 were unable to infect the host cells, and the treated intracellular forms were damaged after 2 h of incubation, when the toxic effects were poorly reverted. New insights on metal toxicity mechanisms are provided, helping to understand how metallic ions influence the parasite's biochemical and physiological processes.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.