Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum.
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The p36/LACK antigen from Leishmania, an analogue of the receptor for activated protein kinase C (PKC), induces high levels of protection against parasite infection in the BALB/c mouse model. This protection is more than twice as high as that elicited by major parasite antigens such as soluble Leishmania antigen or the main surface protease gp63. We have cloned and purified p36/LACK from Leishmania infantum, the causative agent of visceral leishmaniasis in Europe. This protein belongs to the large family of WD 40 repeat proteins confined to eukaryotes and involved in numerous regulatory functions. Differential solubilization and immunofluorescence experiments indicate that p36/LACK is present close to the kinetoplast disc in the cell cytoplasm, probably bound to multiprotein complexes but not to membrane structures. These complexes probably also include cytoplasm PKC isoforms. The use of a genetically-encoded peptide library indicates that p36/LACK binds sequences present in several proteins involved in DNA replication and RNA synthesis. The recognition and binding sequences present in vacuolar proteins and at the beta-chain of major histocompatability complex (MHC) class II suggest the involvement of this regulatory protein in the early mechanisms triggering the protective immune response of the host against the parasite infection. (+info)
Folding stability of the kinetoplastid membrane protein-11 (KMP-11) from Leishmania infantum.
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Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell surface of kinetoplastids, and acts as a potent B- and T-cell immunogen during Leishmania infection. Here we report that the Leishmania infantum KMP-11 secondary structure adopts mainly an alpha-helical conformation at pH 7.5 and that its urea- and thermally-induced unfolding constitute a fully reversible two-step process. This allows estimation of a half-denaturation temperature of approximately 65 degrees C, a delta GDH2O at 20 degrees C of approximately 14.63 kJ.mol-1, and an increment of the reaction heat of approximately 183.92 kJ.mol-1 and an entropy of approximately 543.4 J.mol-1.deg-1, respectively, for the native-denatured equilibrium of the KMP-11 in solution. We also report that the KPM-11 protein is induced to adopt a molten globule state at a pH range between pH 4 and pH 6. As a whole, the stability and the specific features of the denaturing effect induced by changes in pH are similar in KMP-11 to various other lipoproteins. (+info)
Value of Western blotting in the clinical follow-up of canine leishmaniasis.
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Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker. (+info)
Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France.
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Visceral leishmaniosis (VL) due to Leishmania infantum (L. chagasi) is a lethal disease if untreated, but asymptomatic L. infantum infections have been reported previously. A better understanding of parasite transmission, dissemination, and survival in the human host is needed. The purpose of this study was to assess whether L. infantum circulated in peripheral blood of subjects with no history of VL. Sera from 565 blood donors were screened by Western blotting to detect Leishmania-specific antibodies and identify individuals with probable past exposure to Leishmania. Seropositivity was found in 76 donors whose buffy coats were examined by PCR and direct culture. The parasite minicircle kinetoplast DNA was amplified from blood samples of nine donors. Promastigotes were detected by culture in blood samples from nine donors. Only two donors were PCR and culture positive. These results indicate that L. infantum circulates intermittently and at low density in the blood of healthy seropositive individuals, who thus appear to be asymptomatic carriers. Implications for the safety of blood transfusion are discussed. (+info)
High conservation of the fine-scale organisation of chromosome 5 between two pathogenic Leishmania species.
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In a previous work we showed a remarkable conservation of the general structure of the genome (chromo-some number and synteny) among different pathogenic species of Old World Leishmania, indicating the absence of major interchromosomal rearrangements during evolution. In the present study, we have compared the fine structure of chromo-some 5 among two of these divergent species (Leishmania major and Leishmania infantum) by means of physical mapping. Remarkably, the 42 markers jointly mapped on these two chromosomes were found in an identical order along the chromosome. This perfect colinearity of the markers is in striking contrast to the large genetic distance that separates these species and suggests that conservation of the fine-scale organisation of chromosomes may be critical in Leishmania. If this colinearity is confirmed on the whole of the chromosome set, the current systematic sequencing programme of the genome of L.major should greatly help in the development of comparative genetics between different species of Leishmania. (+info)
Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood.
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The PCR technique was applied to the diagnosis of leishmaniasis in dogs, both serologically negative and positive. DNA was taken from lymph node aspirates and blood. The primers 13a and 13b, derived from Leishmania amazonies and Leishmania braziliensis kinetoplast DNA (kDNA), also amplified Leishmania infantum IPT1 constant region of minicircle kDNA. The amplified fragment is 116 bp long. It was cloned and the sequence was determined. A 70-bp inner fragment was designed and used as a probe in dot blot hybridization. A group of 124 dogs was examined, 37 of which showed typical symptoms of disease. PCR was performed on 124 blood samples and 52 lymph node aspirates. Using microscopic examination as the "gold standard," we calculated sensitivity, specificity, and positive and negative predictive values of 100% using lymph node aspirates and values of 85, 80, 95, and 57%, respectively, using blood samples. We found that 40% of the animals without lesions and 38% of the animals with clinical signs gave false-negative results by indirect immunofluorescence antibody testing. These animals could contribute to the spreading of infection among dogs, and represent a potential risk for human health as well. (+info)
Flow cytometric assessment of amphotericin B susceptibility in Leishmania infantum isolates from patients with visceral leishmaniasis.
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Amphotericin B susceptibility was measured by a flow cytometric membrane potential assay in Leishmania infantum promastigotes isolated from 11 immunocompetent children treated with liposomal amphotericin B and 19 HIV-infected young adults treated with intralipid amphotericin B. Susceptibility levels were measured by the 90% inhibitory concentrations (IC90) representing the concentrations of drug that induced a 90% decrease in membrane potential compared with the control culture. In immunocompetent children, treatment was fully effective whatever the susceptibility of isolates to amphotericin B. In immunocompromised adults, on the contrary, unresponsiveness and relapses could be observed in all cases and IC90 increased in the course of successive treatments: a decrease of amphotericin B susceptibility in both promastigote and amastigote forms could be observed in a patient who had six relapses. These results suggest that the success of amphotericin B treatment depends greatly on patient immunity status, and indicate that successive relapses could enhance emergence of amphotericin B resistant isolates. The results demonstrate that the flow cytometric membrane potential assay can be used as an easy and reliable tool for studying the evolution of interactions between amphotericin B and the parasite membrane during long-term treatments. (+info)
Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains.
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A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35 degrees C. The findings open the possibility of using canine peritoneal cells as a model for the screening of leishmanicide drugs and to study the pathogenesis of these species. (+info)