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Frontiers in Immunology 2020Neutrophils act as the first line of defense against invading pathogens. Although traditionally considered in context of their antimicrobial effector functions, the...
Neutrophils act as the first line of defense against invading pathogens. Although traditionally considered in context of their antimicrobial effector functions, the importance of tumor-associated neutrophils (TANs) in the development of cancer has become increasingly clear during the last decade. With regard to their high plasticity, neutrophils were shown to acquire an anti-tumorigenic N1 or a pro-tumorigenic N2 phenotype. Despite the urgent need to get a comprehensive understanding of the interaction of TANs with their tumor microenvironment, most studies still rely on murine tumor models. Here we present for the first time a polarization attempt to generate N1 and N2 neutrophils from primary human neutrophils . Our results underscore that N1-polarized neutrophils have a pro-inflammatory phenotype characterized among others by a higher level of intercellular adhesion molecule (ICAM)-1 and high secretion of interferon (IFN)γ-induced protein 10 (IP-10)/C-X-C motif chemokine 10 (CXCL10) and tumor necrosis factor (TNF). Further, we demonstrate that neutrophils incubated under a tumor-mimicking environment show a high cell surface expression of C-X-C motif chemokine receptor 2 (CXCR2) and secrete high levels of interleukin (IL)-8. These findings suggest that it is feasible to polarize blood-derived primary human neutrophils toward N1- and N2-like phenotypes . Further, we hypothesized that the presence of anti-inflammatory neutrophil phenotype is not a phenomenon limited to cancer but also occurs when neutrophils are infected with intracellular pathogens. Indeed, our findings indicate that N2-polarized neutrophils exert a markedly decreased capacity to kill the protozoan parasite and therefore permit parasite persistence.
Topics: Cell Culture Techniques; Cell Differentiation; Humans; Leishmania donovani; Neutrophils; Phenotype
PubMed: 32411122
DOI: 10.3389/fimmu.2020.00532 -
Microbiology Spectrum Aug 2023Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying...
Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying this excessive production of nonprotective antibodies are still poorly understood. Here, we show that a causative agent of VL, Leishmania donovani, induces CD21-dependent formation of tunneling nanotubule (TNT)-like protrusions in B cells. These intercellular connections are used by the parasite to disseminate among cells and propagate B cell activation, and close contact both among the cells and between B cells and parasites is required to achieve this activation. Direct contact between cells and parasites is also observed , as L. donovani can be detected in the splenic B cell area as early as 14 days postinfection. Interestingly, parasites can also glide from macrophages to B cells via TNT-like protrusions. Taken together, our results suggest that, during infection, B cells may acquire L. donovani from macrophages via TNT-like protrusions, and these connections are subsequently exploited by the parasite to disseminate among B cells, thus propagating B cell activation and ultimately leading to polyclonal B cell activation. Leishmania donovani is a causative agent of visceral leishmaniasis, a potentially lethal disease characterized by strong B cell activation and the subsequent excessive production of nonprotective antibodies, which are known to worsen the disease. How activates B cells is still unknown, particularly because this parasite mostly resides inside macrophages and would not have access to B cells during infection. In this study, we describe for the first time how the protozoan parasite Leishmania donovani induces and exploits the formation of protrusions that connect B lymphocytes with each other or with macrophages and glides on these structures from one cell to another. In this way, B cells can acquire from macrophages and become activated upon contact with the parasites. This activation will then lead to antibody production. These findings provide an explanation for how the parasite may propagate B cell activation during infection.
Topics: Humans; Leishmania donovani; Leishmaniasis, Visceral; Macrophages
PubMed: 37404188
DOI: 10.1128/spectrum.05096-22 -
Molecular Diagnosis & Therapy Aug 2018Visceral leishmaniasis (VL), a deadly parasitic disease, is a major public health concern globally. Countries affected by VL have signed the London Declaration on... (Review)
Review
Visceral leishmaniasis (VL), a deadly parasitic disease, is a major public health concern globally. Countries affected by VL have signed the London Declaration on Neglected Tropical Diseases and committed to eliminate VL as a public health problem by 2020. To achieve and sustain VL elimination, it will become progressively important not to miss any remaining cases in the community who can maintain transmission. This requires accurate identification of symptomatic and asymptomatic carriers using highly sensitive diagnostic tools at the primary health service setting. The rK39 rapid diagnostic test (RDT) is the most widely used tool and with its good sensitivity and specificity is the first choice for decentralized diagnosis of VL in endemic areas. However, this test cannot discriminate between current, subclinical, or past infections and is useless for diagnosis of relapses and as a prognostic (cure) test. Importantly, as the goal of elimination of VL as a public health problem is approaching, the number of people susceptible to infection will increase. Therefore, correct diagnosis using a highly sensitive diagnostic test is crucial for applying appropriate treatment and management of cases. Recent advances in molecular techniques have improved Leishmania detection and quantification, and therefore this technology has become increasingly relevant due to its possible application in a variety of clinical sample types. Most importantly, given current problems in identifying asymptomatic individuals because of poor correlation between the main methods of detection, molecular tests are valuable for VL elimination programs, especially to monitor changes in burden of infection in specific communities. This review provides a comprehensive overview of the available VL diagnostics and discusses the usefulness of molecular methods in the diagnosis, quantification, and species differentiation as well as their clinical applications.
Topics: Humans; Immunoassay; Leishmania donovani; Leishmaniasis, Visceral; Molecular Diagnostic Techniques; Molecular Typing; Parasite Load; Protozoan Proteins; Severity of Illness Index
PubMed: 29922885
DOI: 10.1007/s40291-018-0343-y -
Frontiers in Cellular and Infection... 2020Visceral leishmaniasis (VL) caused by parasites of the complex can be fatal in susceptible individuals. Understanding the interactions between host and pathogen is one... (Review)
Review
Visceral leishmaniasis (VL) caused by parasites of the complex can be fatal in susceptible individuals. Understanding the interactions between host and pathogen is one way to obtain leads to develop better drugs and for vaccine development. In recent years multiple omics-based approaches have assisted researchers to gain a more global picture of this interaction in leishmaniasis. Here we review results from studies using three omics-based approaches to study VL caused by in India: (i) chip-based analysis of single nucleotide variants in the first genome-wide association study of host genetic risk factors for VL, followed by analysis of epitope binding to HLA DRB1 risk versus protective alleles; (ii) transcriptional profiling demonstrating pathways important in Amphotericin B treated compared to active VL cases, including demonstration that anti-interleukin-10 unleashes a storm of chemokines and cytokines in whole blood responses to soluble leishmania antigen in active cases; and (iii) a meta-taxonomic approach based on sequencing amplicons derived from regions of 16S ribosomal RNA (16S rRNA) and 18S rRNA genes that allowed us to determine composition of both prokaryotic and eukaryotic gut microflora in VL cases compared to endemic controls. Overall, our omics-based approaches demonstrate that global analyses of genetic risk factors, host responses to infection, and the interaction between host, parasite and the microbiome can point to the most critical factors that determine the outcome of infection.
Topics: Genome-Wide Association Study; Humans; India; Leishmania donovani; Leishmaniasis, Visceral; RNA, Ribosomal, 16S; Transcriptome
PubMed: 33324576
DOI: 10.3389/fcimb.2020.590888 -
Acta Crystallographica. Section F,... Feb 2018Leishmania is an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme...
Leishmania is an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK from L. donovani was cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed the T to be 317.2 K. Kinetic parameters were obtained by functional characterization of L. donovani RK, and the K values for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space group P6, with unit-cell parameters a = b = 100.25, c = 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (V) of 2.45 Å Da and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.
Topics: Amino Acid Sequence; Crystallization; Crystallography, X-Ray; Leishmania donovani; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 29400319
DOI: 10.1107/S2053230X18000109 -
Antimicrobial Agents and Chemotherapy Dec 2019Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin...
Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMM) was induced in three Nepalese clinical strains of with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC) values for PMM parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage ( < 0.05). This effect was most marked in the Sb-resistant (Sb) PMM clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type ( < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMM strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMM) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMM and Sb PMM). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMM In addition, we found that PMM is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.
Topics: Animals; Antiprotozoal Agents; Drug Resistance; Female; Genomics; Humans; Leishmania donovani; Leishmaniasis, Visceral; Lipidomics; Macrophages; Metabolomics; Mice; Mice, Inbred BALB C; Nepal; Parasitic Sensitivity Tests; Paromomycin; Polymorphism, Genetic
PubMed: 31658971
DOI: 10.1128/AAC.00904-19 -
International Journal For Parasitology.... Aug 2018Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control...
Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (Sb, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.
Topics: Antimony; Antiprotozoal Agents; Drug Resistance, Multiple; Genomics; Leishmania donovani; Leishmania infantum; Leishmania major; Multidrug Resistance-Associated Proteins; Parasitic Sensitivity Tests; Paromomycin; Phenotype; Phosphorylcholine; Transcriptome
PubMed: 29689531
DOI: 10.1016/j.ijpddr.2018.04.002 -
International Journal For Parasitology.... Dec 2017Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at...
Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (K, V, K) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania.
Topics: Amphotericin B; Antiprotozoal Agents; Asparaginase; Drug Resistance; Inhibitory Concentration 50; Kinetics; Leishmania donovani; Leishmaniasis, Visceral; Metabolic Networks and Pathways; Models, Molecular; Protozoan Proteins; Real-Time Polymerase Chain Reaction; Scattering, Small Angle; X-Ray Diffraction
PubMed: 28988014
DOI: 10.1016/j.ijpddr.2017.09.003 -
BMC Infectious Diseases May 2021Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and...
BACKGROUND
Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD.
METHODS
RAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics.
RESULTS
The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein.
CONCLUSIONS
The RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.
Topics: Animals; Base Sequence; China; Cluster Analysis; DNA, Protozoan; Genetic Markers; Genetic Variation; Humans; Leishmania donovani; Leishmaniasis, Visceral; Phylogeny; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique; Species Specificity
PubMed: 34020601
DOI: 10.1186/s12879-021-06163-y -
Frontiers in Cellular and Infection... 2021Glutamine synthetase (GS) is one of the most important metabolic enzymes which catalyzes ligation of glutamate and ammonia to form glutamine. Previous studies from our...
Glutamine synthetase (GS) is one of the most important metabolic enzymes which catalyzes ligation of glutamate and ammonia to form glutamine. Previous studies from our lab had revealed significant differences in parasite and host GS enzyme which warranted us to further work on its relevance in parasite. To analyze glutamine synthetase function in , we generated GS overexpressors and knockout mutants and evaluated their ability to grow in monocyte differentiated macrophage and by infections in BALB/c mice. GS knocked out strain showed significant growth retardation with delayed cell cycle progression and morphological alteration. Null mutants exhibited attenuated infectivity both in and experiments and the effect was reverted back when infected with GS complemented parasites. This indicated that the alterations in phenotype observed were indeed due to GS knockout. GS knockout also made the parasite increasingly sensitive to Miltefosine. Detailed investigation of mode of parasite death upon Miltefosine treatment by dual staining with Annexin-V conjugated FITC and propidium iodide, pointed towards apoptotic or necrotic mode of cell death. This is the first report to confirm that GS is essential for the survivability and infectivity of , and can be exploited as a potential drug-target.
Topics: Animals; Glutamate-Ammonia Ligase; Glutamine; Leishmania donovani; Mice; Mice, Inbred BALB C; Phenotype
PubMed: 33732662
DOI: 10.3389/fcimb.2021.622266