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1.
Eleanor C. Saunders William W. Ng Joachim Kloehn Jennifer M. Chambers Milica Ng Malcolm J. McConville 《PLoS pathogens》2014,10(1)
Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism. 相似文献
2.
Protozoan parasites of the genus Leishmania are important human pathogens that differentiate inside host macrophages into an amastigote life cycle stage. Although this stage causes the pathogenesis of leishmaniasis, only few proteins have been implicated in amastigote intracellular survival. Here we compare morphology, infectivity and protein expression of L. donovani LD1S grown in host free (axenic) culture, or exclusively propagated in infected hamsters, with the aim to reveal parasite traits absent in axenic but selected for in hamster-derived amastigotes through leishmanicidal host activities. Axenic and splenic amastigotes showed a striking difference in virulence and the ability to cause experimental hepato-splenomegaly in infected hamsters. 2D-DIGE analysis revealed statistically significant differences in abundance for 152 spots, with 14 spots showing fivefold or higher abundance in splenic amastigotes. Proteins identified by MS analysis include the anti-oxidant enzyme tryparedoxin peroxidase, and enzymes implicated in protein and amino acid metabolism. Analysis of parasite growth in vitro in minimal medium demonstrated increased survival of hamster-derived compared with axenic parasites under conditions that mimic the nutrient poor, cytotoxic phagolysosome. Thus, our comparative proteomics analysis sheds important new light on the biochemistry of bona fide amastigotes and informs on survival factors relevant for intracellular L. donovani infection. 相似文献
3.
Ralton JE Naderer T Piraino HL Bashtannyk TA Callaghan JM McConville MJ 《The Journal of biological chemistry》2003,278(42):40757-40763
The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages. 相似文献
4.
A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans 总被引:10,自引:0,他引:10
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《The Journal of cell biology》1993,123(3):759-766
The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes. 相似文献
5.
Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha 总被引:51,自引:0,他引:51
S J Green R M Crawford J T Hockmeyer M S Meltzer C A Nacy 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(12):4290-4297
Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites. 相似文献
6.
An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs) by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i) hosting amastigotes of either L. major or L. amazonensis and ii) loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i) entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii) the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii) the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms. 相似文献
7.
Khaddouj Benmoussa Johan Garaude Rebeca Acín-Pérez 《Journal of molecular biology》2018,430(21):3906-3921
Metabolic reprogramming of cells from the innate immune system is one of the most noteworthy topics in immunological research nowadays. Upon infection or tissue damage, innate immune cells, such as macrophages, mobilize various immune and metabolic signals to mount a response best suited to eradicate the threat. Current data indicate that both the immune and metabolic responses are closely interconnected. On account of its peculiar position in regulating both of these processes, the mitochondrion has emerged as a critical organelle that orchestrates the coordinated metabolic and immune adaptations in macrophages. Significant effort is now underway to understand how metabolic features of differentiated macrophages regulate their immune specificities with the eventual goal to manipulate cellular metabolism to control immunity. In this review, we highlight some of the recent work that place cellular and mitochondrial metabolism in a central position in the macrophage differentiation program. 相似文献
8.
Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. The immunosuppressant cyclosporin A (CsA), which inhibits the production of interleukin (IL)-1, IL-2, and interferon-gamma, increased infections 3-fold without affecting expression of the Lsh gene. The objective of this study was to determine how activation of macrophages by lymphokines affects the multiplication and propagation of the parasite within liver macrophages. Susceptible C57BL/6J and resistant C57L/J mice were treated with 200 mg/kg CsA and then infected intravenously with 10(7) amastigotes. Two weeks later macrophages were collected from the liver by perfusion, plated on coverslips, and incubated for 4, 24, and 48 hr. The percentage of infected macrophages and the number of amastigotes/100 cells were determined after staining the cells with Giemsa's stain. The number of infected macrophages and amastigotes per macrophage was significantly greater in animals of both strains that had been treated with CsA. This study demonstrated clearly that lymphokines or other soluble mediators produced by T cells act, in part, to control infection by L. donovani by minimizing both multiplication within macrophages and their dispersion. 相似文献
9.
Sousa-Franco J Araújo-Mendes E Silva-Jardim I L-Santos J Faria DR Dutra WO Horta MF 《Microbes and infection / Institut Pasteur》2006,8(2):390-400
The immune mechanisms that underlie resistance and susceptibility to leishmaniasis are not completely understood for all species of Leishmania. It is becoming clear that the immune response, the parasite elimination by the host and, as a result, the outcome of the disease depend both on the host and on the species of the infecting Leishmania. Here, we analyzed the outcome of the infection of BALB/c mice with L. guyanensis in vivo and in vitro. We showed that BALB/c mice, which are a prototype of susceptible host for most species of Leishmania, dying from these infections, develop insignificant or no cutaneous lesions and eliminate the parasite when infected with promastigotes of L. guyanensis. In vitro, we found that thioglycollate-elicited BALB/c peritoneal macrophages, which are unable to eliminate L. amazonensis without previous activation with cytokines or lipopolysaccharide, can kill L. guyanensis amastigotes. This is the first report showing that infection of peritoneal macrophages with stationary phase promastigotes efficiently triggers innate microbicidal mechanisms that are effective in eliminating the amastigotes, without exogenous activation. We demonstrated that L. guyanensis amastigotes die inside the macrophages through an apoptotic process that is independent of nitric oxide and is mediated by reactive oxygen intermediates generated in the host cell during infection. This innate killing mechanism of macrophages may account for the resistance of BALB/c mice to infection by L. guyanensis. 相似文献
10.
Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages 总被引:2,自引:0,他引:2
Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages of Leishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54-cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1:3, 1:5, and 1:10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasite/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1-5 parasites. After 48- and 72-h exposures, the macrophages harbored 6-11 and 11-20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with amastigote forms, after inoculations of the macrophages cultures with promastigotes at the dilutions previously used for amastigotes, only 48-78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5 X10(6) promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much les virulent for these experimental hosts. 相似文献
11.
Macrophage activation to kill Leishmania tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents 总被引:12,自引:0,他引:12
Resident peritoneal macrophages and macrophages elicited by injection of C3H/HeN mice with sterile inflammatory agents were exposed to amastigotes of Leishmania tropica in vitro and treated with lymphokines. Resident macrophages developed the capacity to kill intracellular parasites; microbicidal activity of activated resident cells ranged between 60 and 80%. In contrast, inflammatory macrophages responded poorly to lymphokines for intracellular killing of amastigotes; microbicidal activity of cells elicited with chronic inflammatory agents ranged between 0 and 45%. Defective intracellular killing of L. tropica by inflammatory macrophages was independent of the agent used to elicit the cells, but was clearly associated with the number of immature macrophages in the population. That intracellular killing capacity may reflect the presence of a killing mechanism in tissue-derived cells that is not yet developed in undifferentiated macrophages is supported by studies with peripheral blood monocytes: these cells were also incapable of eliminating intracellular amastigotes in the presence of potent activating factors. These observations on inflammatory macrophage interactions with amastigotes may provide important insights into the chronic nature of leishmanial disease. 相似文献
12.
Castro R Scott K Jordan T Evans B Craig J Peters EL Swier K 《The Journal of parasitology》2006,92(6):1162-1170
Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes. 相似文献
13.
Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models
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Eleanor C. Saunders Thomas Naderer Jenny Chambers Scott M. Landfear Malcolm J. McConville 《Molecular microbiology》2018,108(2):143-158
Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1‐3 mutant which lacks the major glucose transporters LmxGT1‐3. We identified a novel suppressor line (Δlmxgt1‐3s2) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1‐3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non‐essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources. 相似文献
14.
Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages
The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival. 相似文献
15.
Fernando Real Pilar Tavares Veras Florentino Luiza Campos Reis Eduardo M. Ramos‐Sanchez Patricia Sampaio Tavares Veras Hiro Goto Renato Arruda Mortara 《Cellular microbiology》2014,16(10):1549-1564
The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long‐term‐infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro‐irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non‐irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system. 相似文献
16.
Kilpatrick ED Terajima M Koster FT Catalina MD Cruz J Ennis FA 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(5):3297-3304
Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IkappaBalpha and IkappaBbeta and the related protein NF-kappaB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-kappaB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-kappaB was still observed. Cysteine peptidase inhibitors prevented IkappaBalpha, IkappaBbeta, and NF-kappaB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IkappaBalpha in vitro. LPS-mediated IkappaBalpha and IkappaBbeta degradation was not affected by these inhibitors, confirming that the site of degradation of IkappaBalpha, IkappaBbeta, and NF-kappaB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-kappaB and consequently inhibit IL-12 production. 相似文献
17.
In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages. 相似文献
18.
Khoa D. Tran Danielle P. Vieira Marco A. Sanchez Jessica Valli Eva Gluenz Scott M. Landfear 《PloS one》2015,10(8)
In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δ
kharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δ
kharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δ
kharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δ
kharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis. 相似文献
19.
Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages 总被引:3,自引:0,他引:3
The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes. 相似文献
20.
Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs. 相似文献