The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity.

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4+ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155-/-) mice. Infection was controlled significantly quicker in the miR155-/- mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155-/- mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ+CD8+ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major-infected miR155-/- mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major-infected miR155-/- DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155-/- T cells was significantly enhanced in L. major-infected miR155-/- DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 to increase phagolysosomal iron availability.

Natural resistance-associated macrophage protein 1 (Nramp1) was originally discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal compartments, where it functions as an iron transporter. But the mechanism by which Nramp1 controls host-pathogen dynamics and determines final outcome of an infection is yet to be fully deciphered. Whether the expression of Nramp1 is altered in response to a pathogen attack is also unknown. To address these, Nramp1 status was examined in Leishmania major-infected murine macrophages. We observed that at 12 hrs post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron content and enhanced intracellular parasite growth. Leishmania infection-induced Nramp1 downregulation was caused by ubiquitin-proteasome degradation pathway, which in turn was found to be mediated by the iron-regulatory peptide hormone hepcidin. Blocking of Nramp1 degradation with proteasome inhibitor or transcriptional agonist of hepcidin resulted in depletion of phagolysosomal iron pool that led to significant reduction of intracellular parasite burden. Interestingly, Nramp1 level was restored to normalcy after 30 hrs of infection with a concomitant drop in phagolysosomal iron, which is suggestive of a host counteractive response to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for phagolysosomal iron. We also report Nramp1 as a novel target for hepcidin, and this 'hepcidin-Nramp1' axis may have a broader role in regulating macrophage iron homeostasis.

Leishmania heme uptake involves LmFLVCRb, a novel porphyrin transporter essential for the parasite.

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present.

In Leishmania major, the Homolog of the Oncogene PES1 May Play a Critical Role in Parasite Infectivity.

Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals in control and eradication of this disease. From the parasite genome, we have identified the homologue of the human oncogene PES1 in Leishmania major (LmjPES). It has been demonstrated that PES1 is involved in several processes such as ribosome biogenesis, cell proliferation and genetic transcription. Our phylogenetic studies showed that LmjPES encodes a highly conserved protein containing three main domains: PES N-terminus (shared with proteins involved in ribosomal biogenesis), BRCT (found in proteins related to DNA repair processes) and MAEBL-type domain (C-terminus, related to erythrocyte invasion in apicomplexan). This gene showed its highest expression level in metacyclic promastigotes, the infective forms; by fluorescence microscopy assay, we demonstrated the nuclear localization of LmjPES protein. After generating mutant parasites overexpressing LmjPES, we observed that these clones displayed a dramatic increase in the ratio of cell infection within macrophages. Furthermore, BALB/c mice infected with these transgenic parasites exhibited higher footpad inflammation compared to those inoculated with non-overexpressing parasites.

LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania.

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors' alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica.

Background: Cutaneous leishmaniasis caused by Leishmania species (L. spp) is one of the most important parasitic diseases in humans. To gain information on the metabolite variations and biochemical pathways between L. spp, we used the comparative metabolome of metacyclic promastigotes in the Iranian isolates of L. major and L. tropica by proton nuclear magnetic resonance (1H-NMR). Methods: L. tropica and L. major were collected from three areas of Iran, namely Gonbad, Mashhad, and Bam, between 2017 and 2018, and were cultured. The metacyclic promastigote of each species was separated, and cell metabolites were extracted. 1H-NMR spectroscopy was applied, and the data were processed using ProMatab in MATLAB (version 7.8.0.347). Multivariate statistical analyses, including the principal component analysis and the orthogonal projections to latent structures discriminant analysis, were performed to identify the discriminative metabolites between the two L. spp. Metabolites with variable influences in projection values of more than one and a P value of less than 0.05 were marked as significant differences. Results: A set of metabolites were detected, and 24 significantly differentially expressed metabolites were found between the metacyclic forms of L. major and L. tropica isolates. The top differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased more in L. tropica than L. major (P<0.005), whereas asparagine, 3-hydroxybutyrate, L-proline, and kynurenine were increased significantly in L. major (P<0.01). The significantly altered metabolites were involved in eight metabolic pathways. Conclusion: Metabolomics, as an invaluable technique, yielded significant metabolites, and their biochemical pathways related to the metacyclic promastigotes of L. major and L. tropica. The findings offer greater insights into parasite biology and how pathogens adapt to their hosts.

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

Quantitative proteomic analysis to determine differentially expressed proteins in axenic amastigotes of Leishmania tropica and Leishmania major.

Cutaneous leishmaniasis is commonly caused by Leishmania major and Leishmania tropica. In the present study, the differential expression of proteins was identified in the amastigote-like forms of L. tropica and L. major in Iranian isolates. Initially, the samples were cultured and identified using PCR-RFLP technique. The Leishmania isolates were then grown in host-free (axenic) culture and prepared to amastigote-like forms, followed by the extraction of their proteins. To identify significant differentially expressed proteins (DEPs) of two types of Leishmania, the label-free quantitative proteomic technique was used based on sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 51 up/down-DEPs (fold change >2 and p-value <.05) were identified between the axenic amastigote forms of L. major and L. tropica. Of these, 34 and 17 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process analyses for DEPs; furthermore, the metabolic process and translation were disclosed as top category in the up-regulated proteins of both L. major and L. tropica species. Also, the KEGG analysis revealed carbon metabolism and metabolic pathways term as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Taken together, the numerous novel DEPs identified between the studied species could help fully understand the molecular mechanisms of pathogenesis and provide potential drug targets and vaccine candidates.

Apoptotic blebs from Leishmania major-infected macrophages as a new approach for cutaneous leishmaniasis vaccination.

We focused on apoptotic blebs from Leishmania major-infected macrophages as a vaccine for cutaneous leishmaniasis. Apoptosis was induced in L. major-infected J774A.1 cells in order to prepare apoptotic blebs. Test groups of BALB/c mice were immunized with these at doses of 1 × 106, 5 × 106 or 1 × 107 blebs. An immunization control group received Leishmania lysate antigens. The results showed that as the number of apoptotic bodies increased, the lymphocyte proliferation index increased, and this was proportional to IFN-γ level in the test groups. Additionally, the difference of IFN-γ, IL-4, IFN-γ/IL-4 ratio, or total IgG (p < 0.0001) in all groups was statistically significant compared to the negative control group. The highest IFN-γ (514.0 ± 40.92 pg/mL) and IFN-γ/IL-4 ratio (2.94 ± 0.22) were observed in the group that received 1 × 107 apoptotic blebs. The highest levels of IL-4 (244.6 ± 38.8 pg/mL) and total IgG (5626 ± 377 µg/mL) were observed in the immunization control group. Reflecting these data, no lesions were observed in any of the groups vaccinated with apoptotic blebs after 12 weeks. In summary, the use of apoptotic blebs from L. major-infected macrophages is protective against the challenge with L. major in this animal model.

PAS domain-containing phosphoglycerate kinase deficiency in Leishmania major results in increased autophagosome formation and cell death.

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major.

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5' mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice.

BACKGROUND & OBJECTIVES: Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL), however, treatment failures are frequent. Nimodipine, a calcium channel blocker is known to show promising antiprotozoal effects. Here, we investigated the antileishmanial effect of Nimodipine in both in vitro and in vivo BALB/c mice model of CL. We also compared the in vivo effect with amphotericin B and meglumine antimoniate in the experimental CL mice model. METHODS: Colorimetric alamar blue assay and J774 A.1 mouse macrophage cells were used to determine the effect of nimodipine on promastigotes and amastigotes viability, respectively. Then, the in vivo activity of nimodipine was compared to that of conventional therapies in both the early and established courses of Leishmania major infection in susceptible non-healing BALB/c mice. RESULTS: Nimodipine was highly active against promastigotes and amastigotes of L. major with IC50 values of 49.40 and 15.03 µM, respectively. In the early model, the combination therapy with meglumine antimoniate and nimodipine showed no parasites in the spleen or footpad of animals. The footpad thickness was significantly lower in mice treated with either nimodipine (1 mg/kg or 2.5 mg/kg) or amphotericin B compared to the control group in the established lesions model. However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy). INTERPRETATION & CONCLUSION: The effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models. Since nimodipine is more cost-effective than conventional therapies, our results merit further investigation in other animal models and voluntary human subjects.

Ascorbate-Dependent Peroxidase (APX) from Leishmania amazonensis Is a Reactive Oxygen Species-Induced Essential Enzyme That Regulates Virulence.

The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis, are poorly understood. In L. amazonensis, reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages. Tight spatial and temporal regulation of H2O2 is key to this signaling mechanism, suggesting a role for ascorbate-dependent peroxidase (APX), which degrades mitochondrial H2O2 Earlier studies showed that APX-null L. major parasites are viable, accumulate higher levels of H2O2, generate a greater yield of infective metacyclic promastigotes, and have increased virulence. In contrast, we found that in L. amazonensis, the ROS-inducible APX is essential for survival of all life cycle stages. APX-null promastigotes could not be generated, and parasites carrying a single APX allele were impaired in their ability to infect macrophages and induce cutaneous lesions in mice. Similar to what was reported for L. major, APX depletion in L. amazonensis enhanced differentiation of metacyclic promastigotes and amastigotes, but the parasites failed to replicate after infecting macrophages. APX expression restored APX single-knockout infectivity, while expression of catalytically inactive APX drastically reduced virulence. APX overexpression in wild-type promastigotes reduced metacyclogenesis, but enhanced intracellular survival following macrophage infection or inoculation into mice. Collectively, our data support a role for APX-regulated mitochondrial H2O2 in promoting differentiation of virulent forms in both L. major and L. amazonensis Our results also uncover a unique requirement for APX-mediated control of ROS levels for survival and successful intracellular replication of L. amazonensis.

Leishmania HASP and SHERP Genes Are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host.

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation.

Combined chemotherapy manifest less severe immunopathology effects in helminth-protozoa comorbidity.

BACKGROUND: Co-infection with Leishmania major and Schistosoma mansoni may have significant consequences for disease progression, severity and subsequent transmission dynamics. Pentavalent antimonials and Praziquantel (PZQ) are used as first line of treatment for Leishmania and Schistosoma infections respectively. However, there is limited insight on how combined therapy with the standard drugs impacts the host in comorbidity. The study aimed to determine the efficacy of combined chemotherapy using Pentostam (P) and PZQ in murine model co-infected with L. major and S. mansoni. METHODS: A 3 × 4 factorial design with three parasite infection groups (Lm, Sm, Lm + Sm to represent L. major, S. mansoni and L. major + S. mansoni respectively) and four treatment regimens [P, PZQ, P + PZQ, and PBS designating Pentostam (GlaxoSmithKline UK), Praziquantel (Biltricide®, Bayer Ag. Leverkusen, Germany), Pentostam + Praziquantel and Phosphate buffered saline] as factors was applied. RESULTS: Significant changes were observed in the serum Interferon gamma (IFN-γ), and Macrophage inflammatory protein-one alpha (MIP-1α) levels among various treatment groups between week 8 and week 10 (p < 0.05). There was increased IFN-γ in the L. major infected mice subjected to PZQ and PBS, and in L. major + S. mansoni infected BALB/c mice treated with P + PZQ. Subsequently, MIP-1α levels increased significantly in both the L. major infected mice under PZQ and PBS and in L. major + S. mansoni infected BALB/c mice undergoing concurrent chemotherapy with P + PZQ between 8 and 10 weeks (p < 0.05). In the comorbidity, simultaneous chemotherapy resulted in less severe histopathological effects in the liver. CONCLUSION: It was evident, combined first line of treatment is a more effective strategy in managing co-infection of L. major and S. mansoni. The findings denote simultaneous chemotherapy compliments immunomodulation in the helminth-protozoa comorbidity hence, less severe pathological effects following the parasites infection. Recent cases of increased incidences of polyparasitism in vertebrates call for better ways to manage co-infections. The findings presented necessitate intrinsic biological interest on examining optimal combined chemotherapeutic agents strategies in helminth-protozoa concomitance and the related infections abatement trends vis-a-vis host-parasite relationships.

Cyclic Nucleotide-Specific Phosphodiesterases as Potential Drug Targets for Anti-Leishmania Therapy.

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.

Loss of virulence in NAD(P)H cytochrome b5 oxidoreductase deficient Leishmania major.

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature. The viability assay by flow cytometry as well as microscopic studies suggests that linoleate is an absolute requirement for Leishmania promastigote survival in delipidated media. Western blot analysis suggested that infection with log phase linoleate deficient mutant (KO) results in increased level of NF-κBp65, IκB and IKKß phosphorylation in RAW264.7 cells. Similarly, the log phase KO infected RAW264.7 cells show dramatic increment of COX-2 expression and TNF-α secretion, compared to control or Ncb5or complement (CM) cell lines. The activation of inflammatory signaling pathways by KO mutant is significantly reduced when the RAW264.7 cells are pre-treated with BSA bound linoleate. Together, these findings confirmed that the leishmanial linoleate inhibits both COX-2 and TNF-α expression in macrophage via the inactivation of NF-κB signaling pathway. The stationary phase of KO promastigotes shows avirulence after infection in macrophages as well as inoculation into BALB/c mice; whereas CM cell lines show virulence. Collectively, these data provide strong evidence that de novo linoleate synthesis in Leishmania is an essential for parasite survival at extracellular promastigote stage as well as intracellular amastigote stage.