Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis.

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.

Lipidomic alterations of in vitro macrophage infection by L. infantum and L. amazonensis.

Particular lipid profiles have been found in two different protozoa of the Leishmania genus. Leishmania infantum, a visceral leishmaniasis causative agent and Leishmania amazonensis, a cutaneous leishmaniasis, reveal distinctive lipid contents of phosphatidylethanolamine and phosphatidylserine plasmalogens, sphingolipids, phosphatidylinositols, phosphatidylcholine, and phosphatidylethanolamine, which have been shown to be related to species, life-cycle of the parasite, and macrophage infection. L. infantum displayed a higher content of phosphatidylethanolamine plasmalogens than L. amazonensis, which may help to differentiate their unique clinical manifestations. Phosphatidylserines plasmalogens are also found to be an important lipid class for the intracellular form of the parasite. Our findings also reveal lipid classes that may be involved in visceralization pathways and parasite differentiation.

Role of prostaglandin F2α production in lipid bodies from Leishmania infantum chagasi: insights on virulence.

Lipid bodies (LB; lipid droplets) are cytoplasmic organelles involved in lipid metabolism. Mammalian LBs display an important role in host-pathogen interactions, but the role of parasite LBs in biosynthesis of prostaglandin F2α (PGF2α) has not been investigated. We report herein that LBs increased in abundance during development of Leishmania infantum chagasi to a virulent metacyclic stage, as did the expression of PGF2α synthase (PGFS). The amount of parasite LBs and PGF2α were modulated by exogenous arachidonic acid. During macrophage infection, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). We detected PGF2α receptor (FP) on the Leishmania PV surface. The blockage of FP with AL8810, a selective antagonist, hampered Leishmania infection, whereas the irreversible inhibition of cyclooxygenase with aspirin increased the parasite burden. These data demonstrate novel functions for parasite-derived LBs and PGF2α in the cellular metabolism of Leishmania and its evasion of the host immune response.

Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system.

The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB) using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity - chitosan (Cs) and chondroitin sulfate (ChS) - were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ) nanoparticles, chitosan-chondroitin sulfate (NQC) nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB) nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and a polydispersity index of 0.2. The measured zeta potential of the nanoparticles indicated a positive charge in their surface, while scanning and transmission electron microscopy revealed spherical nanoparticles with a smooth surface. Attenuated total reflectance-Fourier transform infrared spectroscopy analysis showed an electrostatic interaction between the polymers, whereas the release profile of AmpB from the NQC-AmpB nanoparticles showed a controlled release. In addition, the Cs; ChS; and NQ, NQC, and NQC-AmpB nanoparticles proved to be effective against promastigotes of Leishmania amazonensis and Leishmania chagasi, with a synergistic effect observed between Cs and ChS. Moreover, the applied NQ, NQC, and NQC-AmpB compounds demonstrated low toxicity in murine macrophages, as well as null hemolytic activity in type O(+) human red blood cells. Pure AmpB demonstrated high toxicity in the macrophages. The results show that cells infected with L. amazonensis and later treated with Cs, ChS, NQ, NQC, NQC-AmpB nanoparticles, or pure AmpB presented with a significant reduction in parasite number in the order of 24%, 31%, 55%, 66%, 90%, and 89%, respectively. The data presented indicate that the engineered NQC-AmpB nanoparticles could potentially be used as an alternative therapy to treat leishmaniasis, mainly due its low toxicity to mammals' cells.

Turn-on fluorescent probes for nitric oxide sensing based on the ortho-hydroxyamino structure showing no interference with dehydroascorbic acid.

A new family of fluorescent synthetic molecular probes for nitric oxide sensing based on ortho-hydroxyamino-triarylpyrylium salts is presented.

Protein haptenation by amoxicillin: high resolution mass spectrometry analysis and identification of target proteins in serum.

Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including ß-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis.

BACKGROUND: Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals. METHODOLOGY AND PRINCIPAL FINDINGS: VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110-130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients' sera. SIGNIFICANCE: Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.

Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection.

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

Specific serodiagnosis of canine visceral leishmaniasis using Leishmania species ribosomal protein extracts.

In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

Key role of the 3' untranslated region in the cell cycle regulated expression of the Leishmania infantum histone H2A genes: minor synergistic effect of the 5' untranslated region.

BACKGROUND: Histone synthesis in Leishmania is tightly coupled to DNA replication by a post-transcriptional mechanism operating at the level of translation. RESULTS: In this work we have analyzed the implication of the 5' and 3' untranslated regions (UTR) in the cell cycle regulated expression of the histone H2A in Leishmania infantum. For that purpose, L. infantum promastigotes were stably transfected with different plasmid constructs in which the CAT coding region used as a reporter was flanked by the 5' and 3' UTR regions of the different H2A genes. We report that in spite of their sequence differences, histone H2A 5' and 3' UTRs conferred a cell cycle dependent pattern of expression on the CAT reporter since de novo synthesis of CAT increased when parasites enter the S phase. Using one established L. infantum cell line we showed that CAT expression is controlled by the same regulatory events that control the endogenous histone gene expression. Thus, although we did not detect changes in the level of CAT mRNAs during cell cycle progression, a drastic change in the polysome profiles of CAT mRNAs was observed during the progression from G1 to S phase. In the S phase CAT mRNAs were on polyribosomal fractions, but in the G1 phase the association of CAT transcripts with ribosomes was impaired. Furthermore, it was determined that the addition of just the H2A 3' UTR to the CAT reporter gene is sufficient to achieve a similar pattern of post-transcriptional regulation indicating that this region contains the major regulatory sequences involved in the cell cycle dependent expression of the H2A genes. On the other hand, although CAT transcripts bearing the H2A 5' alone were translated both in the G1 and S phase, higher percentages of transcripts were detected on polyribosomes in the S phase correlating with an increase in the de novo synthesis of CAT. Thus, it can be concluded that this region also contributes, although to a minor extent than the 3' UTR, in the enhancement of translation in the S phase relative to the G1 phase. CONCLUSION: Our findings indicate that both, the 5' and the 3' UTRs contain sequence elements that contribute to the cell cycle expression of L. infantum H2A. The 3' UTR region is essential for cell cycle dependent translation of the L. infantum H2A transcripts whereas the 5' UTR has a minor contribution in their S phase dependent translation.

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice.

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.

A DNA aptamer population specifically detects Leishmania infantum H2A antigen.

Aptamers are short single-stranded DNA or RNA oligonucleotides that are selected in vitro by their affinity and specificity for the target. Binding is a consequence of the particular tertiary structure that they are able to acquire, depending on their sequence. Parasites of the genus Leishmania belongs to the lower eukaryote order Kinetoplastida that causes leishmaniosis in man and animals. Histone genes in Leishmania are of considerable interest because these flagellates do not condense their chromatin during mitosis. Thus, the study of the structural features of histones has been considered of particular interest and, as a result, in recent years a great number of histone genes have been characterized in trypanosomatids. Histones are extremely conserved proteins, reflecting their apparent universality of function. Sequence similarity of kinetoplastid core histones those of higher eukaryotes is found predominantly in the globular region with high sequence divergences in the N- and in the C-terminal domains. These divergences indicate that they may be potential diagnostic and/or therapeutics targets. We have successfully isolated a pool of DNA sequences, named SELH2A, which specifically binds to Leishmania infantum H2A. When tested in an enzyme-linked oligonucleotide assay, slot blot and Western blot analysis, the aptamer pool exhibited specificity in its ability to bind only to H2A antigen but not to other proteins from L. infantum including other histones. Thus, it appears that this novel anti-H2A aptamer population may be of potential application as a diagnostic system for leishmaniosis.

The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3'-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 degrees C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 degrees C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 degrees C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 degrees C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 degrees C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 degrees C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the deltaHSP70 mutant synthesizes HSP70 at a lower rate than the wild-type parasites. Overall, our data suggest that the biological function of the HSP70-II gene is to top up HSP70 levels under conditions of stress.

The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation.

BACKGROUND: Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation. RESULTS: Here we report the studies on the expression of the heat shock protein 83 (HSP83) genes of Leishmania infantum. Confirming previous observations for other Leishmania species, we found that the L. infantum HSP83 transcripts also show a temperature-dependent accumulation that is controlled by a post-transcriptional mechanism involving sequences located in the 3'-untranslated region (3'-UTR). However, contrary to that described for L. amazonensis, the accumulation of the HSP83 transcripts in L. infantum is dependent on active protein synthesis. The translation of HSP83 transcripts is enhanced during heat shock and, as first described in L. amazonensis, we show that the 3'-UTR of the L. infantum HSP83 gene is essential for this translational control. Measurement of the steady-state levels of HSP83 transcripts along the promastigote-to-amastigote differentiation evidenced a specific profile of HSP83 RNAs: after an initial accumulation of HSP83 transcripts observed short after (2 h) incubation in the differentiation conditions, the amount of HSP83 RNA decreased to a steady-state level lower than in undifferentiated promastigotes. We show that this transient accumulation is linked to the presence of the 3'-UTR and flanking regions. Again, an 8-fold increase in translation of the HSP83 transcripts is observed short after the initiation of the axenic differentiation, but it is not sustained after 9 h. CONCLUSIONS: This transient expression of HSP83 genes could be relevant for the differentiation of Leishmania, and the underlying regulatory mechanism may be part of the developmental program of this parasite.