-
American Journal of Physiology. Renal... Sep 2014Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a...
Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a transcription factor, NFAT5/TonEBP, because it activates expression of osmoprotective genes betaine/glycine transporter 1 (BGT1) and sodium/myo-inositol transporter (SMIT), and water channel aquaporin-2, all of which are imperative for concentrating urine. Leishmania parasites evade macrophage immune defenses by activating protein tyrosine phosphatases, among which SHP-1 is critical. We previously demonstrated that SHP-1 inhibits tonicity-dependent activation of NFAT5/TonEBP in HEK293 cells through screening a genome-wide small interfering (si) RNA library against phosphatases (Zhou X, Gallazzini M, Burg MB, Ferraris JD. Proc Natl Acad Sci USA 107: 7072-7077, 2010). We sought to examine whether Leishmania can activate SHP-1 and inhibit NFAT5/TonEBP activity in the renal inner medulla in a murine model of visceral leishmaniasis by injection of female BALB/c mice with a single intravenous dose of 5 × 10(5) L. chagasi metacyclic promastigotes. We found that SHP-1 is expressed in the kidney inner medulla. L. chagasi activates SHP-1 with an increase in stimulatory phosphorylation of SHP-1-Y536 in the region. L. chagasi reduces expression of NFAT5/TonEBP mRNA and protein as well as expression of its targeted genes: BGT1, SMIT, and aquaporin-2. The culture supernatant from L. chagasi metacyclic promastigotes increases SHP-1 protein abundance and potently inhibits NFAT5 transcriptional activity in mIMCD3 cells. However, L. chagasi in our animal model has no significant effect on urinary concentration. We conclude that L. chagasi, most likely through its secreted virulence factors, activates SHP-1 and reduces NFAT5/TonEBP gene expression, which leads to reduced NFAT5/TonEBP transcriptional activity in the kidney inner medulla.
Topics: Animals; Aquaporin 2; Cell Line; Disease Models, Animal; Female; GABA Plasma Membrane Transport Proteins; Gene Expression Regulation; Kidney Concentrating Ability; Kidney Cortex; Kidney Medulla; Leishmania infantum; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Protein Tyrosine Phosphatase, Non-Receptor Type 6; STAT3 Transcription Factor; Symporters; Transcription Factors
PubMed: 24990897
DOI: 10.1152/ajprenal.00006.2014 -
Biochimie Oct 2021Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe...
Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC of 1.56 and 1.71 μM, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 μM, while for L. chagasi the values were 2.48, 1.84 and 1.60 μM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis.
Topics: Antiprotozoal Agents; Caffeic Acids; Humans; Leishmania infantum; Leishmaniasis, Visceral; Macrophages; THP-1 Cells
PubMed: 34216704
DOI: 10.1016/j.biochi.2021.06.015 -
Parasites & Vectors Jun 2021Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite...
BACKGROUND
Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite and associated histological changes in the pancreas of dogs are poorly studied. Therefore, the objectives of this study were to evaluate the frequency of detection and load of amastigotes in the pancreas of L. infantum-seropositive dogs and to identify the clinical signs and histological changes associated with parasitism of this organ.
METHODS
One hundred forty-three dogs from an endemic area in Brazil that tested seropositive for L. infantum were studied. The dogs were clinically examined, killed, and necropsied between 2013 and 2014. One fragment of the pancreas was randomly collected for histopathology and immunohistochemistry, and spleen and bone marrow were collected for culture.
RESULTS
Leishmania amastigotes were detected in the pancreas of 22 dogs (15.4%) by immunohistochemistry, all exhibiting L. infantum parasitism in the spleen and/or bone marrow. Poor body condition and cachexia were only associated with infection of the pancreas with Leishmania spp. (p = 0.021) and were found in 40.9% of dogs with pancreatic infection. Anorexia, vomiting, and/or diarrhea were observed in 9.2% of dogs with pancreatitis. The median parasite load in the pancreas was 1.4 infected macrophages/mm. Pancreatic histological changes and their frequencies were: granulomatous pancreatitis (28.0%), lymphoplasmacytic pancreatitis (23.8%), acinar cell degeneration (6.3%), fibrosis (5.6%), hemorrhage (2.1%), eosinophilic pancreatitis (0.7%), suppurative pancreatitis (0.7%), and necrosis (0.7%).
CONCLUSIONS
The present results demonstrate that L. infantum is one of the etiological agents of chronic pancreatitis in dogs; however, the frequency of detection and parasite load are low in this organ. The lack of an association of poor body condition and cachexia with pancreatitis and the low frequency of clinical signs commonly associated with pancreatitis suggest that a significant portion of the organ is not affected by this parasite. On the other hand, the association of poor body condition and cachexia with concomitant infection of the pancreas, spleen, and/or bone marrow with this parasite suggests that these manifestations are the result of a more advanced stage of canine visceral leishmaniasis.
Topics: Animals; Dog Diseases; Dogs; Female; Histological Techniques; Immunohistochemistry; Leishmania infantum; Leishmaniasis, Visceral; Male; Pancreas; Parasite Load
PubMed: 34118967
DOI: 10.1186/s13071-021-04813-3 -
ELife Jan 2022are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are...
are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low-efficiency crosses between two strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the strains and extends to other species, including and , a capacity to generate intra- and interspecific hybrids. Whole-genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen . By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.
Topics: Gene Expression; Genes, Protozoan; Hybridization, Genetic; In Vitro Techniques; Leishmania donovani; Leishmania infantum; Leishmania major; RNA-Seq; Single-Cell Analysis; Stress, Physiological
PubMed: 34994687
DOI: 10.7554/eLife.73488 -
PloS One 2020Leishmania infantum infantum (LII) is one of the species that causes visceral leishmaniasis (VL) in the Old World, while L. infantum chagasi (LIC) is present in the New... (Comparative Study)
Comparative Study
Leishmania infantum infantum (LII) is one of the species that causes visceral leishmaniasis (VL) in the Old World, while L. infantum chagasi (LIC) is present in the New World. Few studies address biological differences or the behavior of these strains during infection. These parasites live inside cells of their hosts, continuously evading microbicidal mechanisms and modulating the immune responses of these cells. One of the mechanisms used by these protozoa involves the L-arginine metabolism. Understanding the differences between Leishmania species and establishing an improved murine model for study of leishmaniasis are matters of extreme importance. Thereby, the objectives of this work were to analyze the biological and molecular differences between two Leishmania infantum strains (LII and LIC) and the degree of susceptibility to infection of mice with different genetic backgrounds. The infectivity in vivo and in vitro of LII and LIC strains was evaluated in BALB/c and Swiss Webster mice, as well the NOS and ARG activities. The LII strain was more infective than the LIC strain both in vivo and in vitro. In animals infected by the LII and LIC strains, differences in NOS and ARG activities occurred. In vitro, promastigotes of LII isolated from BALB/c and Swiss Webster mice showed higher ARG activity than LIC promastigotes during the growth curve. However, no difference was observed in intracellular NO production by promastigotes of these strains. The ARG gene sequences were compared, and those of both strains were identical. However, despite the similarity, the strains showed different expression levels of this gene. It can be concluded that although L. chagasi strains are considered identical to L. infantum strains from a molecular point of view, these strains have different biological behavior.
Topics: Animals; Female; Leishmania infantum; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C
PubMed: 33270636
DOI: 10.1371/journal.pone.0230545 -
Scientific Reports Dec 2017Whole blood stimulation with soluble Leishmania antigen (SLA), followed by plasma cytokine and chemokine determination, provides means of detecting subjects with...
Whole blood stimulation with soluble Leishmania antigen (SLA), followed by plasma cytokine and chemokine determination, provides means of detecting subjects with asymptomatic Leishmania infection. This work examines the potential of Protein Saver 903 cards for the storage and transport of SLA-stimulated dried plasma spot samples. Blood was collected from asymptomatic and negative control subjects living in a Leishmania infantum- (Spain) and Leishmania donovani-endemic area (Bangladesh). After SLA-stimulation, three types of sample were prepared: frozen liquid plasma (-20 °C), and plasma dropped onto Protein Saver cards kept at -20 °C (DPS-FZ), and at ambient temperature (DPS-AT). The concentrations of IFN-γ, IL-2, CXCL10, CXCL9, CCL2 and CXCL8 in the thawed liquid plasma (TLP), DPS-FZ and DPS-AT samples were then determined. Strong correlations were seen between the TLP and DPS-FZ/AT samples for all the studied cytokines/chemokines in both the L. infantum and L. donovani areas. Protein Saver 903 cards would therefore appear to allow for the transport of SLA-stimulated plasma samples by courier at ambient temperature. The CXCL10 and CXCL9 detectable in these plasma spots provided robust markers for identifying asymptomatic subjects from both endemic areas. This easy procedure opens up new possibilities for field studies in resource-limited settings, which could help in Leishmania control.
Topics: Antigens, Protozoan; Asymptomatic Diseases; Chemokines; Dried Blood Spot Testing; Female; Humans; Leishmania donovani; Leishmania infantum; Leishmaniasis, Visceral; Male; Solubility
PubMed: 29222521
DOI: 10.1038/s41598-017-17315-z -
PLoS Neglected Tropical Diseases Feb 2015The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under...
BACKGROUND
The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters.
METHODOLOGY/PRINCIPAL FINDINGS
A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival.
CONCLUSIONS/SIGNIFICANCE
Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.
Topics: Animals; Antimony; Antiprotozoal Agents; Cricetinae; Humans; India; Leishmania infantum; Leishmaniasis, Visceral; Luciferases; Parasite Load; Phosphorylcholine; Recombinant Proteins
PubMed: 25679212
DOI: 10.1371/journal.pntd.0003556 -
Parasites & Vectors Sep 2017Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis...
Sand fly and Leishmania spp. survey in Vojvodina (Serbia): first detection of Leishmania infantum DNA in sand flies and the first record of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908.
BACKGROUND
Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis started to emerge for the first time in Vojvodina Province (previously non-endemic region of Serbia). Reports of introduced, and later on autochthonous cases of leishmaniasis alerted the possibility of disease emergence. The aim of this study was to bridge more than a half a century wide gap in entomological surveillance of sand fly vectors in Vojvodina, as well as to verify the presence of the vector species that could support Leishmania spp. circulation.
RESULTS
During the period 2013-2015, a total of 136 sand flies were collected from 48 of 80 surveyed locations. Four sand fly species of the genus Phlebotomus were detected: P. papatasi, P. perfiliewi, P. mascittii and P. neglectus. Detection of P. mascittii represents the first record of this species for the sand fly fauna in Vojvodina and in Serbia. All female specimens (n = 80) were tested for Leishmania spp. DNA, and three blood-fed P. papatasi specimens were positive (4%). One positive DNA sample was successfully amplified by ITS1 nPCR. The RFLP analysis of the resulting 350 bp fragment showed a typical pattern of L. infantum, and the ITS1 partial sequence blasted in GenBank confirmed 100% identity with L. infantum and L. donovani complex sequences. This result represents the first record of both Leishmania spp. and L. infantum DNA from sand flies in Vojvodina, and in Serbia.
CONCLUSIONS
Presence of autochthonous canine leishmaniasis cases, records of Phlebotomus (Larroussius) species proven vectors of L. infantum (P. perfiliewi and P. neglectus) and detection of L. infantum DNA from wild caught (non-competent) vectors, prove that L. infantum is present in Vojvodina and indicates a probable circulation in the region.
Topics: Animals; Cross-Sectional Studies; Dog Diseases; Dogs; Endemic Diseases; Female; Humans; Insect Vectors; Leishmania infantum; Leishmaniasis, Visceral; Male; Phlebotomus; Phylogeny; Psychodidae; Serbia
PubMed: 28950895
DOI: 10.1186/s13071-017-2386-z -
Revista Brasileira de Parasitologia... 2011Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania)infantum chagasi. Peridomestic sandflies...
Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania)infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L. (L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.
Topics: Alleles; Animals; Dogs; Genes, Protozoan; Leishmania infantum
PubMed: 21439231
DOI: 10.1590/s1984-29612011000100009 -
Experimental Parasitology Nov 2016In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are...
In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.
Topics: Animals; Cell Line; Flow Cytometry; Inhibitory Concentration 50; Leishmania braziliensis; Leishmania donovani; Leishmania infantum; Macrophages; Mice; Microscopy, Electron, Transmission; Quinine
PubMed: 27480054
DOI: 10.1016/j.exppara.2016.07.013