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We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×10(4) promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.  相似文献   

3.
Episomal expression of the major surface glycoprotein (gp63) sense and antisense mRNAs in Leishmania amazonensis was found previously to modulate the expression of this molecule as well as its infection of macrophages in vitro. Here, we evaluated the in vivo infectivity of these transfectants in BALB/c mice. Antisense downregulation of gp63 renders this parasite sensitive to complement-mediated lysis and less infective to mice, as indicated by a delay in lesion development and a significant reduction in lesion size and parasite loads at the site of inoculation and in the draining lymph nodes (DLNs). CD4+ cells at the site of inoculation decreased in number more rapidly and were 2-fold less numerous than those in controls by week 4. The number of IFN-gamma-positive cells was higher, while IL-10 positive cells were undetectable. In DLNs, CD4+ cells were higher in number, and the profile of cytokine-positive cells followed essentially the same patterns--found at the site of inoculation. These results suggest that the downregulation of gp63 increases extracellular lysis of the mutants by complement, in the in vivo environment, and reduces their infection of macrophages, resulting in a type 1 immune response seen at the site of inoculation and DLNs.  相似文献   

4.
Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of "metacyclic" promastigotes in the mouthparts of the vector is discussed.  相似文献   

5.
A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37% (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13% (6/46) of the sand flies that blood fed on dogs in the early stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogs were in an advanced stage of disease. Flagellates developed in 9% (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9% (3/34) of those that fed on hamsters with lesions due to a parasite of the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.  相似文献   

6.
The major zinc metalloprotease of Leishmania (gp63), an important determinant of parasite virulence, is attached to the parasite surface via a glycosylphosphatidylinositol anchor. Here we report the spontaneous release of proteolytically active gp63 from a number of Leishmania isolates, causing cutaneous and visceral disease. To investigate the mechanism(s) of gp63 release, we transfected a gp63-deficient variant of Leishmania amazonensis with constructs expressing gp63 and various mutants thereof. Surprisingly, approximately half of wild type gp63 was found in the culture supernatant 12 h post-synthesis. Biochemical analysis of the extracellular gp63 revealed two forms of the protein, one that is released from the cell surface, and another, that apparently is directly secreted. Release of cell surface gp63 was significantly reduced when the proteolytic activity of the protein was inactivated by site-specific mutagenesis or inhibited by zinc chelation, suggesting that release involves autoproteolysis. The extracellular gp63 does not contain a glycosylphosphatidylinositol moiety or ethanolamine, indicating that phospholipolysis is not involved in the release process. Release of gp63 is also independent of glycosylation. The finding of proteolytically active, extracellular gp63 produced by multiple Leishmania isolates suggests a potential role of the extracellular enzyme in substrate degradation relevant to their survival in both the mammalian host and the insect vector.  相似文献   

7.
Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.  相似文献   

8.
A gene for a Ca2+-transporting ATPase (lmaa1) from the trypanosomatid parasite Leishmania (mexicana) amazonensis was overexpressed in two clones of L. amazonensis differing in their virulence. RNA and protein expression of the gene was increased in transfectants, as was the infectivity of transfectants versus parental types in both mouse and in vitro macrophage infection experiments. The virulence of the almost avirulent clone was enhanced such that it was more virulent than the parental 'virulent' clone. Growth of the parasites in culture as promastigotes, after isolation from mouse lesions, indicated that transfection led to improved survival of promastigotes during the stationary phase of culture. As it is in this culture phase that infective metacyclic forms develop, the key role of the Lmaa1 protein may be in metacyclogenesis. The protein may be important in the synthesis and trafficking of new proteins through the secretory pathway, as we demonstrate, using a green fluorescent protein hybrid and by immunofluorescence, that the Lmaa1 protein is located in the endoplasmic reticulum in promastigotes and amastigotes of L. amazonensis.  相似文献   

9.
Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.  相似文献   

10.
A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.  相似文献   

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