Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available ...regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.
Canine leishmaniasis is a major veterinary issue and also a public health challenge due to its zoonotic potential. In this context, serological evaluation is essential for Canine leishmaniasis ...management. Several serological alternatives, such as rapid diagnostic tests, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT), are well established. In fact, the capacity of distinct tests and antigens, evaluated by their sensitivity and specificity, to detect disease is normally considered sufficient for diagnosing Canine leishmaniasis. In this context, we evaluated the seropositivity using 8 different serological tests (ELISA with Leishmania recombinant proteins (rK39, LicTXNPx); soluble promastigote Leishmania antigens (SPLA); commercial ELISA test) in 82 clinically suspect animals from Northern Portugal. The obtained serological data originated 50% of inconclusive serological information with a mixture of seropositive and seronegative results for individual animals. Cut-off independent risk groups were then generated from the serological data to evaluate the clustering of the samples. This analysis originated risk groups that correlated with the most seropositive samples, suggesting that this method might be used, in a cut-off independent manner, to improve conventional serological evaluation. Ultimately, given that no test prioritization exists, the use of any single serological test increases the potential for misdiagnosis, along with all associated risks for the dog as well as public health. The use of a cut-off independent analysis has the potential to improve the predictive values of these tests, enabling a more accurate evaluation of the dog's condition.
Trypanosomiasis is a parasitic disease affecting both humans and animals
in the form of Human African Trypanosomiasis and Nagana disease,
respectively. Anemia is one of the most common symptoms of
...trypanosomiasis, and if left unchecked can cause severe complications and
even death. Several factors have been associated with the development of
this anemia, including dysregulation of iron homeostasis, but little is known
about the molecular mechanisms involved. Here, using murine models, we
study the involvement of hepcidin, the key regulator of iron metabolism and
an important player in the development of anemia of inflammation. Our
data show two stages for the progression of anemia, to which hepcidin contributes
a first stage when anemia develops, with a likely cytokine-mediated
stimulation of hepcidin and subsequent limitation in iron availability and
erythropoiesis, and a second stage of recovery, where the increase in hepcidin
then declines due to the reduced inflammatory signal and increased
production of erythroid regulators by the kidney, spleen and bone marrow,
thus leading to an increase in iron release and availability, and enhanced erythropoiesis.
In agreement with this, in hepcidin knockout mice, anemia is
much milder and its recovery is complete, in contrast to wild-type animals
which have not fully recovered from anemia after 21 days. Besides all other
factors known to be involved in the development of anemia during trypanosomiasis,
hepcidin clearly makes an important contribution to both its
development and recovery.
A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were ...designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against
and
intracellular amastigotes, against
and against different developmental stages of
. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative
substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two
strains and
strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC
) against THP-1 macrophages ranged between 50 and 100 μM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.
Abstract
Under perturbing conditions such as infection with
Leishmania
, a protozoan parasite living within the phagosomes in mammalian macrophages, cellular and organellar structures, and metabolism ...are dynamically regulated for neutralizing the pressure of parasitism. However, how modulations of the host cell metabolic pathways support
Leishmania
infection remains unknown. Herein, we report that lipid accumulation heightens the susceptibility of mice to
L. donovani
infection and promotes resistance to first‐line anti‐leishmanial drugs. Despite being pro‐inflammatory, the in vitro generated uninfected lipid‐laden macrophages (LLMs) or adipose‐tissue macrophages (ATMs) display lower levels of reactive oxygen and nitrogen species. Upon infection, LLMs secrete higher IL‐10 and lower IL‐12p70 cytokines, inhibiting CD4
+
T cell activation and Th1 response suggesting a key modulatory role for intramacrophage lipid accumulation in anti‐leishmanial host defence. We, therefore, examined this causal relationship between lipids and immunomodulation using an in vivo high‐fat diet (HFD) mouse model. HFD increased the susceptibility to
L. donovani
infection accompanied by a defective CD4
+
Th1 and CD8
+
T cell response. The white adipose tissue of HFD mice displays increased susceptibility to
L. donovani
infection with the preferential infection of F4/80
+
CD11b
+
CD11c
+
macrophages with higher levels of neutral lipids reserve. The HFD increased resistance to a first‐line anti‐leishmanial drug associated with a defective adaptive immune response. These data demonstrate that the accumulation of neutral lipids contributes to susceptibility to visceral leishmaniasis hindering host‐protective immune response and reducing the efficacy of antiparasitic drug therapies.
Abstract
Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for ...several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells
A series of (Z)-2-benzylidenebenzofuran-3-(2H)-ones (aurones) bearing a variety of substituents on rings A and B were synthesized and evaluated for their antiparasitic activity against the ...intracellular amastigote form of Leishmania infantum and their cytotoxicity against human THP1-differentiated macrophages. In general, aurones bearing no substituents on ring A (compounds 4a–4f) exhibit higher toxicity than aurones with 4,6-dimethoxy substitution (compounds 4g–4l). Among the latter, two aurones possessing a 2′-methoxy or a 2′-methyl group (compounds 4i and 4j) exhibit potent antileishmanial activity (IC50=1.3±0.1 μM and IC50=1.6±0.2 μM, resp.), comparable to the activity of the reference drug Amphotericin B, whereas they present significantly lower cytotoxicity than Amphotericin B as deduced by the higher selectivity index.
The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory ...potential. Still, they will act in conjunction with other released parasite-derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non-vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose-dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co-inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose-dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose-dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.
An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and ...highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.
Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.
A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).
Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.
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