ABSTRACT
Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.
Subject(s)
Leishmania major/physiology , Leishmaniasis/immunology , Macrophages/immunology , Nitric Oxide/metabolism , Animals , Cell Growth Processes , Cell Movement , Cell Proliferation , Disease Models, Animal , Host-Pathogen Interactions , Humans , Intravital Microscopy , Mice , Mice, Inbred C57BL , Models, TheoreticalABSTRACT
While the central dogma of molecular biology describes how genetic information flows, gene expression is also affected by epigenetic and epitranscriptomic processes. A recent report by Rajan et al. demonstrates how pseudouridylation of a Leishmania ribosomal rRNA affects the expression of particular proteins: an example of epitranslatomic control.
Subject(s)
Leishmania , RNA, Messenger , Ribosomes , Leishmania/metabolism , Leishmania/genetics , Ribosomes/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , Protein Biosynthesis , RNA, Ribosomal/metabolism , RNA, Ribosomal/geneticsABSTRACT
Recruitment of immune cells with antimicrobial activities is essential to fight local infections but has the potential to trigger immunopathology. Whether the immune system has the ability to sense inflammation intensity and self-adjust accordingly to limit tissue damage remains to be fully established. During local infection with an intracellular pathogen, we have shown that nitric oxide (NO) produced by recruited monocyte-derived cells was essential to limit inflammation and cell recruitment. Mechanistically, we have provided evidence that NO dampened monocyte-derived cell cytokine and chemokine production by inhibiting cellular respiration and reducing cellular ATP:ADP ratio. Such metabolic control operated at the tissue level but only when a sufficient number of NO-producing cells reached the site of infection. Thus, NO production and activity act as a quorum sensing mechanism to help terminate the inflammatory response.
Subject(s)
Cytokines/immunology , Inflammation/immunology , Monocytes/immunology , Nitric Oxide/immunology , Animals , Cells, Cultured , Cytokines/metabolism , HEK293 Cells , Host-Parasite Interactions/immunology , Humans , Inflammation/metabolism , Inflammation/parasitology , Leishmania major/immunology , Leishmania major/physiology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/metabolism , Leishmaniasis, Cutaneous/parasitology , Macrophages/immunology , Macrophages/metabolism , Macrophages/parasitology , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/metabolism , Monocytes/parasitology , Nitric Oxide/metabolism , Quorum Sensing/immunologyABSTRACT
The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.
Subject(s)
Chagas Disease , Extracellular Vesicles , Leishmania , Parasites , Trypanosoma cruzi , Animals , Humans , Chagas Disease/epidemiology , Chagas Disease/parasitologyABSTRACT
Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.
Subject(s)
Leishmania , Phlebotomus , Psychodidae , Animals , Psychodidae/parasitology , Phlebotomus/parasitology , Nutrients , Cell Proliferation , MammalsABSTRACT
Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.
Subject(s)
Leishmania donovani , Psychodidae , Humans , Animals , Oxidative Stress/genetics , DNA Repair/genetics , MutationABSTRACT
Schistosomiasis is a neglected tropical disease caused by the helminth Schistosoma spp. and has the second highest global impact of all parasites. Schistosoma are transmitted through contact with contaminated fresh water predominantly in Africa, Asia, the Middle East, and South America. Due to the widespread prevalence of Schistosoma, co-infection with other infectious agents is common but often poorly described. Herein, we review recent literature describing the impact of Schistosoma co-infection between species and Schistosoma co-infection with blood-borne protozoa, soil-transmitted helminths, various intestinal protozoa, Mycobacterium, Salmonella, various urinary tract infection-causing agents, and viral pathogens. In each case, disease severity and, of particular interest, the immune landscape, are altered as a consequence of co-infection. Understanding the impact of schistosomiasis co-infections will be important when considering treatment strategies and vaccine development moving forward.
Subject(s)
Coinfection , Helminthiasis , Schistosomiasis , Humans , Coinfection/epidemiology , Coinfection/parasitology , Schistosomiasis/complications , Schistosomiasis/epidemiology , Schistosomiasis/parasitology , Africa , Soil/parasitology , Prevalence , Helminthiasis/complications , Helminthiasis/epidemiology , Helminthiasis/parasitologyABSTRACT
Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.
Subject(s)
Actin Cytoskeleton , Actins , Leishmania major , Parasites , Profilins , Animals , Humans , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Actins/chemistry , Actins/metabolism , Amino Acid Motifs , Binding Sites , Conserved Sequence , Crystallization , Crystallography, X-Ray , Leishmania major/cytology , Leishmania major/metabolism , Parasites/cytology , Parasites/metabolism , Profilins/chemistry , Profilins/metabolism , Protein Binding , Protein DomainsABSTRACT
Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.
Subject(s)
Calcineurin , Cyclic AMP , Interleukin-33 , Leishmania donovani , Leishmaniasis, Visceral , Mice, Inbred BALB C , Signal Transduction , Animals , Mice , Calcineurin/metabolism , Cyclic AMP/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/genetics , Interleukin-33/metabolism , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/metabolism , Leishmaniasis, Visceral/parasitology , Macrophages/metabolism , Macrophages/parasitologyABSTRACT
SET domain proteins methylate specific lysines on proteins, triggering stimulation or repression of downstream processes. Twenty-nine SET domain proteins have been identified in Leishmania donovani through sequence annotations. This study initiates the first investigation into these proteins. We find LdSET7 is predominantly cytosolic. Although not essential, set7 deletion slows down promastigote growth and hypersensitizes the parasite to hydroxyurea-induced G1/S arrest. Intriguingly, set7-nulls survive more proficiently than set7+/+ parasites within host macrophages, suggesting that LdSET7 moderates parasite response to the inhospitable intracellular environment. set7-null in vitro promastigote cultures are highly tolerant to hydrogen peroxide (H2O2)-induced stress, reflected in their growth pattern, and no detectable DNA damage at H2O2 concentrations tested. This is linked to reactive oxygen species levels remaining virtually unperturbed in set7-nulls in response to H2O2 exposure, contrasting to increased reactive oxygen species in set7+/+ cells under similar conditions. In analyzing the cell's ability to scavenge hydroperoxides, we find peroxidase activity is not upregulated in response to H2O2 exposure in set7-nulls. Rather, constitutive basal levels of peroxidase activity are significantly higher in these cells, implicating this to be a factor contributing to the parasite's high tolerance to H2O2. Higher levels of peroxidase activity in set7-nulls are coupled to upregulation of tryparedoxin peroxidase transcripts. Rescue experiments using an LdSET7 mutant suggest that LdSET7 methylation activity is critical to the modulation of the cell's response to oxidative environment. Thus, LdSET7 tunes the parasite's behavior within host cells, enabling the establishment and persistence of infection without eradicating the host cell population it needs for survival.
Subject(s)
Leishmania donovani , Oxidative Stress , Peroxidases , Protozoan Proteins , Animals , Hydrogen Peroxide/metabolism , Leishmania donovani/genetics , Leishmania donovani/metabolism , Peroxidases/genetics , Peroxidases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Reactive Oxygen Species/metabolism , PR-SET DomainsABSTRACT
Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell. In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of Leishmania major and showed prolonged expression of IL-1ß at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing toward pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells, we characterized the BLaER1 monocytes as a model for L. major infection. We found that BLaER1 monocytes support infection and activation by Leishmania parasites to the same extent as primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild-type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.
Subject(s)
Inflammasomes , Leishmania , Humans , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Pyroptosis/physiology , Phosphate-Binding Proteins/metabolism , Macrophages , Leishmania/metabolism , Interleukin-1beta/metabolismABSTRACT
Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.
Subject(s)
Leishmania , Metalloendopeptidases , Protozoan Proteins , Virulence Factors , Animals , CRISPR-Cas Systems , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Golgi Matrix Proteins/metabolism , Golgi Matrix Proteins/genetics , Leishmania/metabolism , Leishmania/genetics , Macrophages/parasitology , Macrophages/metabolism , Metalloendopeptidases/metabolism , Metalloendopeptidases/genetics , Protein Transport , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Virulence Factors/metabolism , Virulence Factors/geneticsABSTRACT
Leishmania spp. are obligate intracellular parasites that must be internalized by phagocytic cells to evade immune responses and cause disease. The uptake of both Leishmania promastigotes (insect-stage parasites) and amastigotes (proliferative-stage parasites in humans and mice) by phagocytes is thought to be mainly host cell driven, not parasite driven. Our previous work indicates that host Src- and Abl-family kinases facilitate Leishmania entry into macrophages and pathogenesis in murine cutaneous leishmaniasis. Here, we demonstrate that host spleen tyrosine kinase (SYK) is required for efficient uptake of Leishmania promastigotes and amastigotes. A Src-family kinase-Abl-family kinase-SYK signaling cascade induces Leishmania amastigote internalization. Finally, lesion size and parasite burden during Leishmania infection is significantly decreased in mice lacking SYK in monocytes or by treatment with the SYK inhibitor entospletinib. In summary, SYK is required for maximal Leishmania uptake by macrophages and disease in mice. Our results suggest potential for treating leishmaniasis using host cell-directed agents.
Subject(s)
Leishmania , Leishmaniasis , Parasites , Humans , Animals , Mice , Syk Kinase , Phagocytosis , Leishmaniasis/parasitology , MacrophagesABSTRACT
Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.
Subject(s)
Leishmania major , Proteome , Transcriptome , Leishmania major/metabolism , Leishmania major/genetics , Proteome/metabolism , Humans , RNA Polymerase III/metabolism , RNA Polymerase III/genetics , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Gene Expression RegulationABSTRACT
Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.
Subject(s)
Leishmania , RNA Cap-Binding Proteins/metabolism , Histones/metabolism , Proteomics , RNA, Messenger/metabolism , Cell Cycle , Protein BindingABSTRACT
Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.
Subject(s)
Leishmania , Humans , Leishmania/genetics , Antimony , Chromosomes , AneuploidyABSTRACT
Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection, resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.
Subject(s)
Antimicrobial Cationic Peptides , Cathelicidins , Cyclic AMP Response Element Modulator , Leishmania donovani , Leishmaniasis, Visceral , Macrophages , Mice, Inbred BALB C , Leishmania donovani/drug effects , Animals , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Cationic Peptides/pharmacology , Humans , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/drug therapy , Mice , Macrophages/parasitology , Macrophages/metabolism , THP-1 Cells , Cyclic AMP Response Element Modulator/metabolism , Cyclic AMP Response Element Modulator/genetics , Receptors, Calcitriol/metabolism , Receptors, Calcitriol/genetics , Antiprotozoal Agents/pharmacology , FemaleABSTRACT
BACKGROUND: The mechanisms that mediate immune protection in individuals with subclinical (SC) or asymptomatic infection with L. braziliensis are largely unknown. Neutrophils (PMNs) have been implicated in progressive symptomatic cutaneous leishmaniasis (CL), but their potential participation in maintenance of subclinical infection is unexplored. The aim of this study was to compare the phenotypic and functional profiles of PMNs in individuals with SC infection versus patients with symptomatic CL due to L. braziliensis. METHODS: Subjects were recruited in the endemic region of Corte de Pedra, Bahia, Brazil. Surface markers to define activation status were characterized by flow cytometry. Functional responses of PMNs including phagocytic capacity, production of oxidative species, and oxidative killing of intracellular parasites were studied in vitro. RESULTS: PMNs from individuals with SC infection displayed a more activated phenotype and greater ability to control the infection than PMNs from patients with CL. In contrast, PMNs from patients with CL exhibited higher expression of HLA-DR and higher production of oxidative species than PMNs from subjects with SC infection. CONCLUSION: PMNs from individuals with SC infection can control the infection more efficiently than PMNs from patients with CL, despite the lower production of oxidants. Our observations suggest that L. braziliensis may evade microbicidal mechanisms of PMNs from patients with CL, contributing to parasite dissemination and the establishment of disease.
ABSTRACT
Leishmania parasites are heavily dependent on efficient iron acquisition from a tightly regulated host iron pool for survival and virulence. Prior studies uncovered multiple strategies adopted by the parasite to hijack the iron-regulatory network of macrophages. Despite these extensive studies with infected macrophages, there is limited knowledge of the effect of Leishmania infection on systemic iron homeostasis. This issue is particularly relevant for Leishmania major, which causes localized skin infection with minimal lymphatic spread. We show for the first time that L. major infection in the mouse footpad induced influx of iron at the site of infection through blood with simultaneous upregulation of transferrin receptor 1 and downregulation of phagolysosomal iron exporter Nramp1 expression in the footpad tissue. Interestingly, localized L. major infection had far-reaching effects beyond the infection site triggering anemia-like symptoms. This was evident from depleted physiological iron stores from the liver and bone marrow as well as reduced hemoglobin levels and deformed erythrocytes. The infected mice also developed splenomegaly with signs of splenic stress erythropoiesis as indicated by upregulation of several erythroid-related genes. These observations prompted us to provide oral iron supplementations to the L. major-infected mice, which resulted in a drastic reduction of the parasite load and restoration of iron homeostasis.
Subject(s)
Homeostasis , Iron , Leishmaniasis, Cutaneous , Animals , Mice , Dietary Supplements , Erythrocytes/metabolism , Iron/administration & dosage , Iron/metabolism , Leishmania major , Leishmaniasis, Cutaneous/metabolismABSTRACT
Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.