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The Journal of Parasitology Dec 2012Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon...
Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHS-exposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of complement proteins (including C5 and C9) that form the lytic membrane attack complex.
Topics: Animals; Blotting, Western; Complement System Proteins; Cricetinae; Flow Cytometry; Humans; Leishmania infantum; Mesocricetus; Microscopy, Fluorescence
PubMed: 22662870
DOI: 10.1645/GE-3130.1 -
Communications Biology Jan 2021Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid...
Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control.
Topics: Brazil; DNA, Protozoan; Evolution, Molecular; Gene Deletion; Genes, Protozoan; Genome, Protozoan; Leishmania infantum; Leishmaniasis, Visceral; Molecular Epidemiology; Phylogeny; Sequence Deletion; Whole Genome Sequencing
PubMed: 33514858
DOI: 10.1038/s42003-021-01658-5 -
Bioorganic & Medicinal Chemistry Nov 2014In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim...
In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.
Topics: Animals; Antiprotozoal Agents; Cell Line; Eugenol; Humans; Leishmania infantum; Leishmaniasis, Visceral; Macrophages; Male; Mice; Mice, Inbred BALB C; Thymol
PubMed: 25281268
DOI: 10.1016/j.bmc.2014.08.020 -
Revista Do Instituto de Medicina... 2014Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector...
Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.
Topics: Animals; Cell Movement; Cricetinae; Disease Models, Animal; Exudates and Transudates; Female; Host-Parasite Interactions; Leishmania infantum; Leishmaniasis, Visceral; Male; Psychodidae; Saliva
PubMed: 24553604
DOI: 10.1590/S0036-46652014000100003 -
Brazilian Journal of Medical and... Jun 2007Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL...
Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.
Topics: Animals; Disease Models, Animal; Glomerular Mesangium; Glomerulonephritis; Leishmania infantum; Leishmaniasis, Visceral; Male; Mice; Mice, Inbred BALB C; Time Factors
PubMed: 17581681
DOI: 10.1590/s0100-879x2007000600011 -
Journal of Vector Borne Diseases Dec 2010Kala-azar is the visceral and most severe form of leishmaniasis that leads to death if untreated. The causative agents of visceral leishmaniasis (VL) are members of...
BACKGROUND & OBJECTIVES
Kala-azar is the visceral and most severe form of leishmaniasis that leads to death if untreated. The causative agents of visceral leishmaniasis (VL) are members of Leishmania (L.) donovani complex which includes L. chagasi and L. infantum. Genome sequences have raised the question whether L. chagasi and L. infantum are synonymous or different. This question has important implications for clinical and epidemiological studies, evaluation of vaccines and drugs, and disease control. LCR1 is an immunogenic molecule discovered from L. chagasi with potential as a component of a Leishmania subunit vaccine. If this protein has potentials for being used in a vaccine or diagnostic testing, there should be little variability in this molecule between L. infantum isolates from diverse geographic regions. The aim of this study was to determine whether lcr1 of an Iranian strain of L. infantum was identical to lcr1 of both L. infantum strain from a different geographic region (Spain) and that of an L. chagasi isolate from Brazil.
METHODS
L. infantum isolated from an Iranian kala-azar patient was studied. Lcr1 from this isolate was PCR amplified, cloned, and studied by restriction digest analysis and sequencing.
RESULTS
The sequences of lcr1 of the Iranian L. infantum were completely identical at nucleotide level to lcr1 sequences of both the Spanish L. infantum and the Brazilian L. chagasi strains.
CONCLUSION
Complete conservation of the DNA sequence encoding for LCR1 molecule between geographically distinct Leishmania species adds credibility to the potential for LCR1 as a component of a subunit vaccine and diagnostic test for kala-azar.
Topics: Antigens, Protozoan; Base Sequence; Brazil; Evolution, Molecular; Humans; Iran; Leishmania; Leishmania infantum; Leishmaniasis; Molecular Sequence Data; Protozoan Proteins; Spain
PubMed: 21178212
DOI: No ID Found -
PLoS Neglected Tropical Diseases Jul 2020Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania...
BACKGROUND
Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection.
METHODOLOGY/PRINCIPAL FINDINGS
Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory.
CONCLUSIONS/SIGNIFICANCE
Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiology, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts.
Topics: Animals; Dog Diseases; Dogs; Humans; Italy; Leishmania infantum; Leishmaniasis, Visceral
PubMed: 32706789
DOI: 10.1371/journal.pntd.0008465 -
BMC Molecular Biology Feb 2005The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have...
BACKGROUND
The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells.
RESULTS
The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis.
CONCLUSIONS
The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.
Topics: Animals; Blotting, Northern; Blotting, Western; Cysteine Endopeptidases; DNA Mutational Analysis; Gene Expression; Genes, Protozoan; Humans; Leishmania infantum; Macrophages; Proteins; Protozoan Proteins; RNA, Antisense; RNA, Messenger; Recombinant Proteins; Survival; U937 Cells
PubMed: 15691375
DOI: 10.1186/1471-2199-6-3 -
Anais Brasileiros de Dermatologia 2020American cutaneous leishmaniasis is an infectious dermatosis caused by protozoa of the genus Leishmania, which comprises a broad spectrum of clinical manifestations...
BACKGROUND
American cutaneous leishmaniasis is an infectious dermatosis caused by protozoa of the genus Leishmania, which comprises a broad spectrum of clinical manifestations depending on the parasite species involved in the infections and the immunogenetic response of the host. The use of techniques for amplification of the parasites DNA based on polymerase chain reaction polymerase chain reaction and the recent application of combined techniques, such as high-resolution DNA dissociation, have been described as a viable alternative for the detection and identification of Leishmania spp. in biological samples.
OBJECTIVES
To identify the Leishmania species using the polymerase chain reaction high-resolution DNA dissociation technique in skin biopsies of hospital-treated patients, and compare with results obtained by other molecular identification techniques.
METHODS
A retrospective study assessing patients with suspected American cutaneous leishmaniasis seen at a hospital in São Paulo/Brazil was conducted. The paraffin blocks of 22 patients were analyzed by polymerase chain reaction high-resolution DNA dissociation to confirm the diagnosis and identify the species.
RESULTS
Of the 22 patients with suspected American cutaneous leishmaniasis, the parasite was identified in 14, comprising five cases (35.6%) of infection by L. amazonensis, four (28.5%) by L. braziliensis, two (14.4%) by L. amazonensis+L. infantum chagasi, two (14.4%) by L. guyanensis, and one (7.1%) by Leishmania infantum chagasi. In one of the samples, in which the presence of amastigotes was confirmed on histopathological examination, the polymerase chain reaction high-resolution DNA dissociation technique failed to detect the DNA of the parasite.
STUDY LIMITATIONS
The retrospective nature of the study and small number of patients.
CONCLUSIONS
The method detected and identified Leishmania species in paraffin-embedded skin biopsies with a sensitivity of 96.4% and could be routinely used in the public health system.
Topics: Brazil; Humans; Leishmania; Leishmania infantum; Leishmaniasis, Cutaneous; Leishmaniasis, Mucocutaneous; Retrospective Studies; United States
PubMed: 32518010
DOI: 10.1016/j.abd.2020.02.003 -
Microbes and Infection Nov 2010Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select...
Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4(+) and CD8(+) T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.
Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Foot; Leishmania infantum; Leishmania mexicana; Leishmaniasis; Leishmaniasis Vaccines; Macrophages; Mice; Mice, Inbred BALB C; Nitric Oxide; Protozoan Proteins; Ribosomal Proteins; Saponins
PubMed: 20601076
DOI: 10.1016/j.micinf.2010.06.008