Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

Sphingolipid Degradation in Leishmania (Leishmania) amazonensis

Background

Human leishmaniasis is caused by more than 20 Leishmania species and has a wide range of symptoms. Our recent studies have demonstrated the essential role of sphingolipid degradation in the virulence of Leishmania (Leishmania) major, a species responsible for localized cutaneous leishmaniasis in the Old World. In this study, we investigated the function of sphingolipid degradation in Leishmania (Leishmania) amazonensis, an etiological agent of localized and diffuse cutaneous leishmaniasis in South America.

Methodology/Principal Findings

First, we identified the enzyme LaISCL which is responsible for sphingolipid degradation in L. amazonensis. Primarily localized in the mitochondrion, LaISCL shows increased expression as promastigotes progress from replicative log phase to non-replicative stationary phase. To study its function, null mutants of LaISCL (Laiscl⁻) were generated by targeted gene deletion and complemented through episomal gene add-back. In culture, loss of LaISCL leads to hypersensitivity to acidic pH and poor survival in murine macrophages. In animals, Laiscl⁻ mutants exhibit severely attenuated virulence towards C57BL6 mice but are fully infective towards BALB/c mice. This is drastically different from wild type L. amazonensis which cause severe pathology in both BALB/c and C57BL 6 mice.

Conclusions/Significance

A single enzyme LaISCL is responsible for the turnover of sphingolipids in L. amazonensis. LaISCL exhibits similar expression profile and biochemical property as its ortholog in L. major. Deletion of LaISCL reduces the virulence of L. amazonensis and the outcome of Laiscl⁻-infection is highly dependent on the host''s genetic background. Therefore, compared to L. major, the role of sphingolipid degradation in virulence is substantially different in L. amazonensis. Future studies may reveal whether sphingolipid degradation is required for L. amazonensis to cause diffuse cutaneous infections in humans.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.     

Different Leishmania species determine distinct profiles of immune and histopathological responses in CBA mice

Most experimental studies on leishmaniasis compare two different inbred strains of mice that are resistant or susceptible to one species of Leishmania. In the present study we characterized some cytokines and nitric oxide production as well as histological changes related to resistance and susceptibility in isogenic CBA mice infected with Leishmania major or Leishmania amazonensis. CBA mice are capable of controlling infection with L.  major, but they succumb to infection with L. amazonensis. Cells from susceptible L. amazonensis-infected CBA mice produced interleukin (IL)-4 and IL-10 but no interferon (IFN)-γ. On the other hand, resistant L. major-infected CBA mice produced IFN-γ and IL-10, but IL-4 was detected only in the first week of infection. Histopathological studies showed patterns of tissue responses at the site of the infection and in the draining lymph nodes that correlated with resistance or susceptibility. Resistant mice showed a mixed inflammatory cell infiltration and granulomas in the lesions, whereas in susceptible mice only heavily parasitized macrophages were seen. Our results indicate an important role of the parasite species in determining the pattern of immune response. L. amazonensis induces a Th2-type immune response, whereas L.  major induces a Th1-type response. These factors must be identified and taken into account in the strategies for the development of vaccines against leishmaniasis. The model presented here will be useful for the study of such factors.     

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis

Background

Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil.

Methodology/Principal Findings

Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50 = 2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50 = 128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 = 1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity.

Conclusions/Significance

The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.     

In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis

Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.     

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Background

Three major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures.

Methods/Principal Findings

High resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 µl DNA sample, i.e., less than a single parasite per reaction.

Conclusions/Significance

This new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys.     

Influence of long-term treatment with pravastatin on the survival, evolution of cutaneous lesion and weight of animals infected by Leishmania amazonensis

The high toxicity of current drugs for treatment of leishmaniasis is a major hindrance for controlling the disease. Pravastatin is a well-known drug with anti-inflammatory and immunomodulatory properties that may modulate host defense mechanisms against Leishmania. We evaluated the influence of prolonged pravastatin treatment on the survival of Leishmania amazonensis-infected animals (BALB/c, C57BL6 mice and Syrian hamsters), including weekly measurement of cutaneous lesions (footpad thickness) and weight. Pravastatin improved survival of Leishmania-infected BALB/c mice but not of infected C57BL6 mice or hamsters. On the 50th week of follow-up, 71% of pravastatin-treated Leishmania-infected BALB/c mice were alive against 29% of control group (p < 0.01). Low footpad thickness was found on BALB/c pravastatin treated mice from the 14th week (p < 0.05), and 20th week onward for C57BL6 treated mice. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL6 mice and Syrian hamsters, but not infected BALB/c mice. Our results points to beneficial effects of pravastatin on the evolution of the disease in the murine leishmaniasis model.     

Tamoxifen is effective in the treatment of Leishmania amazonensis infections in mice

Background

Chemotherapy is still a critical issue in the management of leishmaniasis. Until recently, pentavalent antimonials, amphotericin B or pentamidine compounded the classical arsenal of treatment. All these drugs are toxic and have to be administered by the parenteral route. Tamoxifen has been used as an antiestrogen in the treatment and prevention of breast cancer for many years. Its safety and pharmacological profiles are well established in humans. We have shown that tamoxifen is active as an antileishmanial compound in vitro, and in this paper we analyzed the efficacy of tamoxifen for the treatment of mice infected with Leishmania amazonensis, an etiological agent of localized cutaneous leishmaniasis and the main cause of diffuse cutaneous leishmaniasis in South America.

Methodology/Principal Findings

BALB/c mice were infected with L. amazonensis promastigotes. Five weeks post-infection, treatment with 15 daily intraperitoneal injections of 20 mg/kg tamoxifen was administered. Lesion and ulcer sizes were recorded and parasite burden quantified by limiting dilution. A significant decrease in lesion size and ulcer development was noted in mice treated with tamoxifen as compared to control untreated animals. Parasite burden in the inoculation site at the end of treatment was reduced from 10^8.5±0.7 in control untreated animals to 10^5.0±0.0 in tamoxifen-treated mice. Parasite load was also reduced in the draining lymph nodes. The reduction in parasite number was sustained: 6 weeks after the end of treatment, 10^15.5±0.5 parasites were quantified from untreated animals, as opposed to 10^5.1±0.1 parasites detected in treated mice.

Conclusions/Significance

Treatment of BALB/c mice infected with L. amazonensis for 15 days with tamoxifen resulted in significant decrease in lesion size and parasite burden. BALB/c mice infected with L. amazonensis represents a model of extreme susceptibility, and the striking and sustained reduction in the number of parasites in treated animals supports the proposal of further testing of this drug in other models of leishmaniasis.     

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

An In Vitro Model of Antibody-Enhanced Killing of the Intracellular Parasite Leishmania amazonensis

Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.     

Complete assembly,annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.     

The role of the P2X7 receptor in murine cutaneous leishmaniasis: aspects of inflammation and parasite control

Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The immunopathology of leishmaniasis caused by L. amazonensis infection is dependent on the pathogenic role of effector CD4⁺ T cells. Purinergic signalling has been implicated in resistance to infection by different intracellular parasites. In this study, we evaluated the role of the P2X7 receptor in modulating the immune response and susceptibility to infection by L. amazonensis. We found that P2X7-deficient mice are more susceptible to L. amazonensis infection than wild-type (WT) mice. P2X7 deletion resulted in increased lesion size and parasite load. Our histological analysis showed an increase in cell infiltration in infected footpads of P2X7-deficient mice. Analysis of the cytokine profile in footpad homogenates showed increased levels of IFN-γ and decreased TGF-β production in P2X7-deficient mice, suggesting an exaggerated pro-inflammatory response. In addition, we observed that CD4⁺ and CD8⁺ T cells from infected P2X7-deficient mice exhibit a higher proliferative capacity than infected WT mice. These data suggest that P2X7 receptor plays a key role in parasite control by regulating T effector cells and inflammation during L. amazonensis infection.     

Comparative in vivo expression of amastigote up regulated Leishmania genes in three different forms of Leishmaniasis

In Old World Leishmania infections in India, Leishmania donovani is responsible for visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) while L. tropica is responsible for cutaneous leishmaniasis (CL) in humans. The molecular differences between the two species of Leishmania and within the same species causing distinct pathologies that govern the outcome of infection and pathogenesis in the human host are unknown. Quantitative expression of selected genes was evaluated directly in lesion tissues of VL, PKDL and CL patients. Assessment of in vivo mRNA level highlighted substantial differences in gene expression patterns, providing an indication of the genes involved in pathogenesis in the three different forms of Leishmaniasis.     

E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) of Leishmania amazonensis inhibits macrophage activation

Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A_2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.