Lipophosphoglycan Research Articles

Activation Pathways of Murine Macrophages by Lipophosphoglycan from Strains of Leishmania major (FV1 and LV39).

Lipophosphoglycan (LPG) is an important Leishmania virulence factor. It is the most abundant surface glycoconjugate in promastigotes, playing an important role in the interaction with phagocytic cells. While LPG is known to modulate the macrophage immune response during infection, the activation mechanisms triggered by this glycoconjugate have not been fully elucidated. This work investigated the role that LPGs purified from two strains of Leishmania major (FV1 and LV39) play in macrophage activation, considering the differences in their biochemical structures. Bone marrow-derived macrophages from BALB/c mice were stimulated with 10 μg/mL purified LPG from the LV39 and FV1 strains. We then measured the production of nitric oxide (NO) and cytokines, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the activation of MAPK pathways. LPG from the LV39 strain, which has longer poly-galactosylated side chains, induced a more pro-inflammatory profile than that from the FV1 strain. This included higher production of NO, TNF-α, and PGE2, and increased expression of COX-2 and iNOS. Additionally, the phosphorylation of ERK-1/2 and JNK was elevated in macrophages exposed to LPG from the LV39 strain. No difference in IL-10 production was observed in cells stimulated by both LPG. Thus, intraspecific structural differences in LPG contribute to distinct innate immune responses in macrophages.

α-Selective Solid-Phase Synthesis of Glycosyl Phosphate Repeating Structure Via the Phosphoramidite Method.

Lipophosphoglycans (LPGs) are found on the surface of Leishmania, a protozoan parasite, and are immunologically important. Herein, disaccharide 1-phosphate repeating units of LPGs were successfully synthesized on a solid support with high anomeric purity using a disaccharide α-1-phosphoramidite building block. To enhance solubility in the reaction solvent, hydroxy-protecting groups in the form of para-t-butylbenzoyl were introduced to the building block. The saccharide chain was elongated via stable glycosyl boranophosphate linkages, followed by the conversion of inter-sugar linkages to phosphodiester counterparts using an oxaziridine derivative. The addition of a silylating reagent post-reaction with the oxaziridine derivative efficiently facilitated the conversion of boranophosohodiesters to phosphodiesters. This method enabled the α-selective synthesis of up to 15 repeating units, marking the longest homogeneous repeating units of LPGs synthesized chemically. Given the chain length equivalence to native LPGs, the method developed herein holds promise for advancing anti-Leishmanial pharmaceuticals and vaccines.

Molecular regulation of NLRP3 inflammasome activation during parasitic infection.

Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis, and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium, Leishmania, Toxoplasma gondii, Entamoeba histolytica, Trypanosoma cruzi, and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites' growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome's action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.

Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition.

Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Leishmania surface molecule lipophosphoglycan-TLR2 interaction moderates TPL2-mediated TLR2 signalling for parasite survival.

Leishmania donovani, a protozoan parasite, resides and replicates in macrophages and inflicts the potentially fatal disease visceral leishmaniasis (VL). The parasite-expressed surface lipophosphoglycan (LPG) was implicated in binding TLR2 on NK cells, but the modus operandi of its disease-promoting influence remained unknown. As TPL2, a member of the MAPK module in mammalian macrophages, was implicated in the anti-inflammatory immune response and promoting pathogen survival, we investigated the possibility of TPL2-directed LPG-TLR2 signalling in Leishmania infection. We observed that TLR2 or TPL2 blockade differentially influenced the TLR2 ligand proteoglycan (PGN)-induced p38MAPK and ERK-1/2 activation. TLR2 blockade abrogated the PGN-induced TPL2 activation. L. donovani infection impaired the Akt activation whereas, upon TPL2 inhibition, the infection fails to control Akt phosphorylation. In L. donovani-infected macrophages, TLR2 blocking negatively affected p38, Akt and TPL2 phosphorylation while ERK1/2 phosphorylation increased relative to the infection alone. TPL2 blockade reduced TGF-β, but increased TNF-α expression and diminished amastigote count in macrophages. While exploring stimulation patterns of TLR2 ligands, LPG, unlike PGN, selectively increased TLR2 expression in macrophages. LPG blockade increased p38MAPK and AKT, but slightly affected ERK-1/2 and significantly reduced TPL2 phosphorylation from L. donovani-infected macrophages. Molecular docking and molecular dynamics analysis drew a parallel between LPG's glycan chain lengths with the frequency of interaction with TLR2 which might impact TLR2 signalling. Therefore, the parasite regulates the TLR2 signalling via TPL2 when elicited by LPG-TLR2 interaction for pathogenesis.

Leishmania major-derived lipophosphoglycan influences the host's early immune response by inducing platelet activation and DKK1 production via TLR1/2.

Platelets are rapidly deployed to infection sites and respond to pathogenic molecules via pattern recognition receptors (TLR, NLRP). Dickkopf1 (DKK1) is a quintessential Wnt antagonist produced by a variety of cell types including platelets, endothelial cells, and is known to modulate pro-inflammatory responses in infectious diseases and cancer. Moreover, DKK1 is critical for forming leukocyte-platelet aggregates and induction of type 2 cell-mediated immune responses. Our previous publication showed activated platelets release DKK1 following Leishmania major recognition. Here we probed the role of the key surface virulence glycoconjugate lipophosphoglycan (LPG), on DKK1 production using null mutants deficient in LPG synthesis (Δlpg1- and Δlpg2-). Leishmania-induced DKK1 production was reduced to control levels in the absence of LPG in both mutants and was restored upon re-expression of the cognate LPG1 or LPG2 genes. Furthermore, the formation of leukocyte-platelet aggregates was dependent on LPG. LPG mediated platelet activation and DKK1 production occurs through TLR1/2. Thus, LPG is a key virulence factor that induces DKK1 production from activated platelets, and the circulating DKK1 promotes Th2 cell polarization. This suggests that LPG-activated platelets can drive innate and adaptive immune responses to Leishmania infection.

C-reactive protein binds to short phosphoglycan repeats of Leishmania secreted proteophosphoglycans and activates complement.

Human C-reactive protein (CRP) binds to lipophosphoglycan (LPG), a virulence factor of Leishmania spp., through the repeating phosphodisaccharide region. We report here that both major components of promastigote secretory gel (PSG), the filamentous proteophosphoglycan (fPPG) and the secreted acid phosphatase (ScAP), are also ligands. CRP binding was mainly associated with the flagellar pocket when LPG deficient Leishmania mexicana parasites were examined by fluorescent microscopy, consistent with binding to secreted material. ScAP is a major ligand in purified fPPG from parasite culture as demonstrated by much reduced binding to a ScAP deficient mutant fPPG in plate binding assays and ligand blotting. Nevertheless, in sandfly derived PSG fPPG is a major component and the major CRP binding component. Previously we showed high avidity of CRP for LPG ligand required multiple disaccharide repeats. ScAP and fPPG only have short repeats but they retain high avidity for CRP revealed by surface plasmon resonance because they are found in multiple copies on the phosphoglycan. The fPPG from many species such as L. donovani and L. mexicana bound CRP strongly but L. tropica and L. amazonensis had low amounts of binding. The extent of side chain substitution of [-PO4-6Galβ1-4Manα1-] disaccharides correlates inversely with binding of CRP. The ligand for the CRP on different species all had similar binding avidity as the half maximal binding concentration was similar. Since the PSG is injected with the parasites into host blood pools and phosphoglycans (PG) are known to deplete complement, we showed that CRP makes a significant contribution to the activation of complement by PSG using serum from naive donors.

Inflammatory responses during trichomoniasis: The role of Toll-like receptors and inflammasomes.

Toll-like receptors (TLRs) and inflammasomes belong to the pattern recognition receptors (PRRs) of innate immunity identifying conserved compounds produced by pathogens or discharged by injured cells. Different cell subsets in the human urogenital system, such as epithelial cells and infiltrating leukocytes, express different kinds of TLRs (such as TLR2, TLR3, TLR4, TLR5 and TLR9) as well as inflammasomes (such as NLRP3, NLRC4 and AIM2). Various types of the Trichomonas vaginalis-derived components such as glycosyl-phosphatidylinositol (GPI), T. vaginalis virus (TVV), Lipophosphoglycan (LPG) and flagellin can be recognized by TLR2, TLR3, TLR4 and TLR5, respectively, leading to the production of proinflammatory cytokines and chemokines in the cervicovaginal mucosa. The T. vaginalis-induced inflammasomes can lead to pyroptosis as well as the release of IL-1β and IL-18 promoting innate and adaptive immune responses. The PRR-mediated responses to T. vaginalis may contribute to the induction of protective immune responses, local inflammation, promotion of co-infections, or even the development of malignancies, for example, prostate cancer. The protective or pathogenic roles of the TLRs and inflammasomes during trichomoniasis are highlighted in this review. A better understanding of PRR-mediated responses provides invaluable insights to develop effective immunotherapeutic strategies against T. vaginalis infection.

Leishmania species: A narrative review on surface proteins with structural aspects involved in host-pathogen interaction.

In tropical and subtropical regions of the world, leishmaniasis is endemic and causes a range of clinical symptoms in people, from severe tegumentary forms (such as cutaneous, mucocutaneous, and diffuse leishmaniasis) to lethal visceral forms. The protozoan parasite of the genus Leishmania causes leishmaniasis, which is still a significant public health issue, according to the World Health Organization 2022. The public's worry about the neglected tropical disease is growing as new foci of the illness arise, which are exacerbated by alterations in behavior, changes in the environment, and an enlarged range of sand fly vectors. Leishmania research has advanced significantly during the past three decades in a few different avenues. Despite several studies on Leishmania, many issues, such as illness control, parasite resistance, parasite clearance, etc., remain unresolved. The key virulence variables that play a role in the pathogenicity-host-pathogen relationship of the parasite are comprehensively discussed in this paper. The important Leishmania virulence factors, such as Kinetoplastid Membrane Protein-11 (KMP-11), Leishmanolysin (GP63), Proteophosphoglycan (PPG), Lipophosphoglycan (LPG), Glycosylinositol Phospholipids (GIPL), and others, have an impact on the pathophysiology of the disease and enable the parasite to spread the infection. Leishmania infection may arise from virulence factors; they are treatable with medications or vaccinations more promptly and might greatly shorten the duration of treatment. Additionally, our research sought to present a modeled structure of a few putative virulence factors that might aid in the development of new chemotherapeutic approaches for the treatment of leishmaniasis. The predicted virulence protein's structure is utilized to design novel drugs, therapeutic targets, and immunizations for considerable advantage from a higher understanding of the host immune response.

Leishmania LPG interacts with LRR5/LRR6 of macrophage TLR4 for parasite invasion and impairs the macrophage functions.

Visceral leishmaniasis (VL) is a severe form of leishmaniasis, primarily affecting the poor in developing countries. Although several studies have highlighted the importance of toll-like receptors (TLRs) in the pathophysiology of leishmaniasis, the role of specific TLRs and their binding partners involved in Leishmania donovani uptake are still elusive. To investigate the mechanism of L. donovani entry inside the macrophages, we found that the parasite lipophosphoglycan (LPG) interacted with the macrophage TLR4, leading to parasite uptake without any significant alteration of macrophage cell viability. Increased parasite numbers within macrophages markedly inhibited lipopolysachharide-induced pro-inflammatory cytokines gene expression. Silencing of macrophage-TLR4, or inhibition of parasite-LPG, significantly stemmed parasite infection in macrophages. Interestingly, we observed a significant enhancement of macrophage migration, and generation of reactive oxygen species (ROS) in the parasite-infected TLR4-silenced macrophages, whereas parasite infection in TLR4-overexpressed macrophages exhibited a notable reduction of macrophage migration and ROS generation. Moreover, mutations in the leucine-rich repeats (LRRs), particularly LRR5 and LRR6, significantly prevented TLR4 interaction with LPG, thus inhibiting cellular parasite entry. All these results suggest that parasite LPG recognition by the LRR5 and LRR6 of macrophage-TLR4 facilitated parasite entry, and impaired macrophage functions. Therefore, targeting LRR5/LRR6 interactions with LPG could provide a novel option to prevent VL.

Trypanosoma brucei Lipophosphoglycan Activates Host Immune Responses via the TLR-mediated p38 MAP Kinase and NF-κB Pathways

Objective: This study was aimed at investigating the immunoregulatory effects of trypanosomal lipophosphoglycan (LPG) anchored to trypanosome membranes, including the formation of neutrophil extracellular traps (NETs) and neutrophil cytokine release after parasite infection. The interaction of cell surface TLR receptors with LPG, which signals cellular responses during Trypanosma brucei infection, was systematically investigated. Methods: The cytokine expression profile in neutrophils after exposure to T. brucei LPG, and the involvement of TLR2, TLR4, p38 MAP kinase, and NF-κB in NET formation were studied with molecular immunological approaches including quantitative PCR, western blotting and immunofluorescence. Results: T. brucei-derived LPG induced phosphorylation of p38 MAP kinase and NF-κB, thereby stimulating neutrophil secretion of IL-1β, IL-8, and TNF-α. The blockade of Toll-like receptor 2/4 and specific inhibitors of MyD88, p38 MAP kinase, and NF-κB decreased cytokine release and the phosphorylation of both kinases. Furthermore, the exposure of neutrophils containing LPG to IL-1β and LPG-induced cell supernatants promoted the release of NETs. Conclusion: Our findings suggest that T. brucei LPG activates neutrophil IL-1β secretion via the TLR-mediated p38 MAP kinase and NF-κB pathways, thereby promoting the formation of LPG-stimulated NETs.

Leishmania mexicana Lipophosphoglycan Activates Dermal γδ T Cells with Participation of TLR2.

Leishmania transmission by sand flies is detected by dermal cells that recognize ligands, such as lipophosphoglycan (LPG) on the promastigote glycocalyx. Resident dermal cells include γδ T cells, that recognize antigens by TCR or innate receptors, such as TLRs. We analyzed the response of dermal γδ T cells to Leishmania mexicana infections or inoculation of LPG, and whether parasite LPG activates γδ T cells through TLR2. We stimulated γδ T cells with LPG and analyzed colocalization of LPG and TLR2 by confocal microscopy. Activation of TLR2 was evaluated by IκBα phosphorylation. BALB/c mice were inoculated with L. mexicana or LPG in the dermis of earlobes, and LPG+ TLR2+ γδ T cells were analyzed by flow cytometry. TNF+ γδ T cells were examined in earlobe dermis by confocal microscopy. Stimulation with purified LPG showed activation of TLR2 with IκBα phosphorylation in γδ T cells. Inoculation of L. mexicana parasites or LPG into earlobe dermis showed co-expression of LPG+ and TLR2+ in γδ T cells, demonstrating their interaction during infections. A subset of γδ T cells (LPG+ and TLR2-) provided evidence that additional receptors recognize LPG. Inoculation of LPG enhanced overall γδ T cell numbers, including those expressing TNF, whereas infection with the parasite mostly enhanced γδ T cells expressing TNF. L. mexicana LPG is a ligand recognized by TLR2 on γδ-T cells leading to their activation, although contribution of other receptors cannot be ruled out and need to be analyzed to elucidate their contribution during Leishmania infections.

Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types.

Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental 'landmarks' delineating different stages of amastigogenesis qualitatively or quantitatively, including new uses of amastigote-specific markers that showed interesting cellular localizations at the anterior or posterior ends. We compared amastigogenesis in synchronous infections of peritoneal and bone-marrow derived macrophages (PEM, BMM) or dendritic cells (BMDC). Overall, the marker suite expression showed an orderly transition post-infection with similar kinetics between host cell types, with the emergence of several amastigote traits within 12 hours, followed by parasite replication after 24 hours, with parasites in BMM or BMDC initiating DNA replication more slowly. Lipophosphoglycan (LPG) is a Leishmania virulence factor that facilitates metacyclic establishment in host cells but declines in amastigotes. Whereas LPG expression was lost by parasites within PEM by 48 hours, >40% of the parasites infecting BMM or BMDC retained metacyclic-level LPG expression at 72 hr. Thus L. major may prolong LPG expression in different intracellular environments, thereby extending its efficacy in promoting infectivity in situ and during cell-to-cell transfer of parasites expressing this key virulence factor.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 μM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Biochemical Properties of Parasite Virulence Factor: Lesson Learned from Leishmania

Leishmania, a parasitic protozoan, a single-celled organism of the genus trypanosomes that are responsible for the disease leishmaniasis. Transmission occured by sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World. Globally, at least 93 sandfly species are proven or probable vectors. Their primary hosts are vertebrates; Leishmania commonly infects hyraxes, canids, rodents, and humans. Leishmaniasis encompasses diverse clinical syndromes, including cutaneous, mucosal, and potentially life-threatening visceral forms. Three widely known virulence factors belongs to the genus Leishmania include the active compound named proteophosphoglycan (PPG), GP63 metalloprotease and lipophosphoglycan (LPG). these substance established on the surface of the parasite. The aim of this review article is to make an insight of the biochemical characteristics of Leishmania spp virulence factors, the armamentarium that predispose their pathogenesis, its invasion and virulence to the mammalian host.

Effect of hybridization on lipophosphoglycan expression in Leishmania major.

Leishmania major is the causative agent of cutaneous leishmaniasis. It is one of the most studied Leishmania species not only during vector interaction but also in the vertebrate host. Lipophosphoglycan (LPG) is the Leishmania multifunctional virulence factor during host-parasite interaction, whose polymorphisms are involved in the immunopathology of leishmaniasis. Although natural hybrids occur in nature, hybridization of L. major strains in the laboratory was successfully demonstrated. However, LPG expression in the hybrids remains unknown. LPGs from parental (Friedlin, Fn and Seidman,Sd) and hybrids (FnSd3, FnSd4A, FnSd4B, and FnSd6F) were extracted, purified, and their repeat units analyzed by immunoblotting and fluorophore-assisted carbohydrate electrophoresis. Parental strains have distinct profiles in LPG expression, and a mixed profile was observed for all hybrids. Variable levels of NO production by macrophages were detected after LPG exposure (parental and hybrids) and were strain specific.

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite–vector–host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

Lipophosphoglycan From Dermotropic New World Leishmania Upregulates Interleukin-32 and Proinflammatory Cytokines Through TLR4 and NOD2 Receptors.

Interleukin-32 (IL-32) is produced during Leishmania infection, but the components of the parasite that induce its production are unknown. An important multivirulence factor of Leishmania spp. protozoa is the lipophosphoglycan (LPG), which plays a crucial role in the host-parasite interaction. Here, the ability of LPGs from two dermotropic Leishmania species to induce IL-32 production was evaluated in human peripheral blood mononuclear cells (PBMCs). Additionally, the potential receptors involved in this activation were assessed. PBMCs from healthy individuals were stimulated with LPGs from L. amazonensis (La) or L. braziliensis (Lb), live promastigotes of La or Lb and E. coli lipopolysaccharide (LPS, TLR4 agonist) as control. Blockers of TLR4 (Bartonella quintana LPS or monoclonal antibody) and Ponatinib (RIPK2 inhibitor, NOD2 pathway) were used to evaluate the receptors. ELISA was performed for IL-32 expression and cytokine (IL-1β and IL-6) production in cell lysates and in supernatants, respectively. Expression of TLR4 (2 h, 24 h) was assessed by flow cytometry. IL-32γ mRNA transcript was analyzed by qPCR. It was observed that LPG from Leishmania, like whole parasites, induced the production of IL-32, IL-1β and IL-6. Both LPGs induced the expression of IL32γ mRNA. The production of IL-32 was earlier detected (6 h) and positively associated with the production of IL-1β and IL-6. The induction of cytokines (IL-32, IL-1β and IL-6) was dependent on TLR4 and NOD2. The TLR4 was internalized after interaction with LPG. Therefore, our data suggest that LPGs from La and Lb are components of Leishmania able to upregulate IL-32 and other pro-inflammatory cytokines in a TLR4- and NOD2-dependent manner. In addition, LPG-induced IL-32 seems to be necessary for IL-1β and IL-6 production. To identify the parasite factors and host receptors involved in IL-32 induction is crucial to reveal potential targets for novel strategies to control leishmaniasis.

A Ferrier glycosylation/cis-dihydroxylation strategy to synthesizeLeishmaniaspp. lipophosphoglycan-associated βGal(1,4)Man disaccharide

The Galβ(1→4)Man disaccharide, found in the cell surface lipophosphoglycan (LPG) of Leishmania species, has been synthesized by a Ferrier glycosylation/cis-dihydroxylation strategy. This stereoselective method proved efficient for synthesizing the target saccharide in good yield. In addition, we prepared two clickable O-glycoside and phospho-glycoside versions of Galβ(1→4)Man to enable conjugation to protein carriers for further immunological and antibody-binding studies.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.