Simvastatin Resistance of Leishmania amazonensis Induces Sterol Remodeling and Cross-Resistance to Sterol Pathway and Serine Protease Inhibitors.

The sterol biosynthesis pathway of Leishmania spp. is used as a pharmacological target; however, available information about the mechanisms of the regulation and remodeling of sterol-related genes is scarce. The present study investigated compensatory mechanisms of the sterol biosynthesis pathway using an inhibitor of HMG-CoA reductase (simvastatin) and by developing drug-resistant parasites to evaluate the impact on sterol remodeling, cross-resistance, and gene expression. Simvastatin-resistant L. amazonensis parasites (LaSimR) underwent reprogramming of sterol metabolism manifested as an increase in cholestane- and stigmastane-based sterols and a decrease in ergostane-based sterols. The levels of the transcripts of sterol 24-C-methyltransferase (SMT), sterol C14-α-demethylase (C14DM), and protease subtilisin (SUB) were increased in LaSimR. LaSimR was cross-resistance to ketoconazole (a C14DM inhibitor) and remained sensitive to terbinafine (an inhibitor of squalene monooxygenase). Sensitivity of the LaSimR mutant to other antileishmanial drugs unrelated to the sterol biosynthesis pathway, such as trivalent antimony and pentamidine, was similar to that of the wild-type strain; however, LaSimR was cross-resistant to miltefosine, general serine protease inhibitor N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), subtilisin-specific inhibitor 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-(3R)-3-pyrrolidinyl-benzamide dihydrochloride (PF-429242), and tunicamycin. The findings on the regulation of the sterol pathway can support the development of drugs and protease inhibitors targeting this route in parasites.

Virola oleifera-capped gold nanoparticles showing radical-scavenging activity and low cytotoxicity.

The development of effective nanoparticle therapeutics has been hindered by their surface characteristics, such as hydrophobicity and charge. Therefore, the success of biomedical applications with nanoparticles is governed by the control of these characteristics. In this article, we report an efficient green capping method for gold nanoparticles (AuNPs) by a reduction with sodium citrate and capping with Virola oleifera (Vo), which is a green exudate rich in polyphenols and flavonoids. The Vo-capped AuNPs were characterized by UV, DLS, FTIR, Raman, TEM, DPPH, FRAP and their cytotoxicity was evaluated on the viability of Murine macrophage cell. The AuNPs had an average particle size of 15 nm and were stable over a long time, as indicated by their unchanged SPR and zeta potential values. These nanoparticles were assessed for their antioxidant potential using DPPH and FRAP and demonstrated the highest antioxidant activities and low cytotoxicity. We propose that the Virola oleifera-capped AuNPs have potential biomedical applications.

Intranasal vaccination with extracellular serine proteases of Leishmania amazonensis confers protective immunity to BALB/c mice against infection.

BACKGROUND: Previously, we demonstrated that unlike subcutaneous or intramuscular vaccination, intranasal vaccination of BALB/c mice with whole Leishmania amazonensis antigens leads to protection against cutaneous leishmaniasis. Here, the role of parasite serine proteases in the protective immunity was investigated. FINDINGS: Serine Proteases were partially purified from both soluble (LaSP-Sol) and extracellular (LaSP-Ex) Leishmania amazonensis promastigote extracts by aprotinin-agarose chromatography. BALB/c mice were intranasally immunized with LaSP-Sol and LaSP-Ex prior to infection with L. amazonensis. LaSP-Ex but not LaSP-Sol vaccination led to significantly smaller lesions and parasite burdens as compared with non-vaccinated controls. Protection was accompanied by systemic Th1 polarization with increased IFN-γ and decreased IL-4 and IL-10 splenic production. Likewise, increased production of IFN-γ, IL-12 and IL-4 concomitant with decreased TGF-ß and TNF-α was locally observed in the infected footpad. CONCLUSION: This study indicates that extracellular serine proteases of L. amazonensis are strong candidates for a more defined intranasal vaccine against cutaneous leishmaniasis.

Peripheral expression of LACK-mRNA induced by intranasal vaccination with PCI-NEO-LACK defines the protection duration against murine visceral leishmaniasis.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in the protozoan of the genus Leishmania, which is associated with the immunopathogenesis and susceptibility of BALB/c mice to Leishmania major infection. We previously demonstrated that intranasal immunization with a plasmid DNA encoding the p36/LACK leishmanial antigen (pCI-neo-LACK) followed by challenge 7 days after a booster dose effectively protects BALB/c mice against both cutaneous and visceral leishmaniasis. In the present study, the correlation between systemic mRNA expression after nasal DNA uptake, and the duration of protective immunity was addressed. LACK mRNA transcripts were detected in the spleen, brain, cervical lymph nodes and popliteal lymph nodes as early as 7 days, lasting 3 months after vaccination with pCI-neo-LACK. The kinetics of transcript expression correlated with enhanced cutaneous hypersensitivity against parasite antigens. Leishmania chagasi infection at 7 days or 3 months, but not 6 months after vaccination resulted in significantly lower parasite loads as compared with non-vaccinated controls. Protection also correlated with enhanced spleen cell responsiveness to parasite antigens leading to increased IFN- γ and IL-4 and decreased IL-10 production. Together, these data demonstrate that the protection conferred by the intranasal DNA vaccine lasts at least 3 months and is associated with expression of vaccine mRNA in peripheral organs.

Intranasal immunization with LACK-DNA promotes protective immunity in hamsters challenged with Leishmania chagasi.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in protozoans of the genus Leishmania which is associated with the immunopathogenesis and susceptibility of BALB/c mice to L. major infection. Previously, we demonstrated that intranasal immunization with a plasmid carrying the LACK gene of Leishmania infantum (LACK-DNA) promotes protective immunity in BALB/c mice against Leishmania amazonensis and Leishmania chagasi. In the present study, we investigated the protective immunity achieved in hamsters intranasally vaccinated with 2 doses of LACK-DNA (30 µg). Compared with controls (PBS and pCI-neo plasmid), animals vaccinated with LACK-DNA showed significant reduction in parasite loads in the spleen and liver, increased lymphoproliferative response and increased nitric oxide (NO) production by parasite antigen-stimulated splenocytes. Furthermore, hamsters vaccinated with LACK-DNA presented high IgG and IgG2a serum levels when compared to control animals. Our results showed that intranasal vaccination with LACK-DNA promotes protective immune responses in hamsters and demonstrated the broad spectrum of intranasal LACK-DNA efficacy in different host species, confirming previous results in murine cutaneous and visceral leishmaniasis.

Oligopeptidase B from Leishmania amazonensis: molecular cloning, gene expression analysis and molecular model.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. A complete open reading frame of oligopeptidase B from Leishmania amazonensis was amplified with polymerase chain reaction with gradient annealing temperatures using primers designed for the oligopeptidase B gene from L. major. The 2,196-bp fragment coded for a protein of 731 amino acids with a predicted molecular mass of 83.49 KDa. The encoded protein (La_OpB) shares a 90% identity with oligopeptidases of L. major and L. infantum, 84% with L. braziliensis, and approximately 62% identity with Trypanosoma peptidases. The oligopeptidase B gene is expressed in all cycle stages of L. amazonensis. The three dimensional model of La_OpB was obtained by homology modeling based on the structure of prolyl oligopeptidases. We mapped a La_OpB model that presents a greater negative charge than prolyl oligopeptidases; our results suggest a difference in the S2 subsite when compared to oligopeptidases B from Trypanosoma and bacterial oligopeptidases B. The La_OpB model serves as a starting point for its exploration as a potential target source for a rational chemotherapy.