scholarly journals Comparisons of Mutants Lacking the Golgi UDP-Galactose or GDP-Mannose Transporters Establish that Phosphoglycans Are Important for Promastigote but Not Amastigote Virulence in Leishmania major

2007 ◽  
Vol 75 (9) ◽  
pp. 4629-4637 ◽  
Author(s):  
Althea A. Capul ◽  
Suzanne Hickerson ◽  
Tamara Barron ◽  
Salvatore J. Turco ◽  
Stephen M. Beverley
Keyword(s):  
Golgi Apparatus ◽  
Protective Immunity ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Macrophage Infection ◽  
Nucleotide Sugar ◽  
Infectious Cycle ◽  
Null Mutants

ABSTRACT Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(β1,4)Man(α1-PO4)]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2 − L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2 −, instead resembling the L. major lipophosphoglycan-deficient lpg1 − mutant. Metacyclic lpg5A −/lpg5B − promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A − /lpg5B − mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2 − Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

scholarly journals Langerhans cells are negative regulators of the anti-Leishmania response

2011 ◽  
Vol 208 (5) ◽  
pp. 885-891 ◽  
Author(s):  
Kordula Kautz-Neu ◽  
Madelon Noordegraaf ◽  
Stephanie Dinges ◽  
Clare L. Bennett ◽  
Dominik John ◽  
...  
Keyword(s):  
Langerhans Cells ◽  
Protective Immunity ◽  
Low Dose ◽  
Leishmania Major ◽  
Interferon Γ ◽  
Cell Ablation ◽  
Selective Depletion ◽  
T Cell Immune Responses

Migratory skin dendritic cells (DCs) are thought to play an important role in priming T cell immune responses against Leishmania major, but DC subtypes responsible for the induction of protective immunity against this pathogen are still controversial. In this study, we analyzed the role of Langerin+ skin-derived DCs in the Leishmania model using inducible in vivo cell ablation. After physiologically relevant low-dose infection with L. major (1,000 parasites), mice depleted of all Langerin+ DCs developed significantly smaller ear lesions with decreased parasite loads and a reduced number of CD4+ Foxp3+ regulatory T cells (T reg cells) as compared with controls. This was accompanied by increased interferon γ production in lymph nodes in the absence of Langerin+ DCs. Moreover, selective depletion of Langerhans cells (LCs) demonstrated that the absence of LCs, and not Langerin+ dermal DC, was responsible for the reduced T reg cell immigration and the enhanced Th1 response, resulting in attenuated disease. Our data reveal a unique and novel suppressive role for epidermal LCs in L. major infection by driving the expansion of T reg cells. A better understanding of the various roles of different DC subsets in cutaneous leishmaniasis will improve the development of a potent therapeutic/prophylactic vaccine.

scholarly journals Leishmania major MPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage

2009 ◽  
Vol 9 (1) ◽  
pp. 22-30 ◽  
Author(s):  
Miguel A. Morales ◽  
Pascale Pescher ◽  
Gerald F. Späth
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
De Novo ◽  
Parasite Growth ◽  
Growth Defect ◽  
Protein Kinase Activity ◽  
Macrophage Infection ◽  
Infectious Cycle

ABSTRACT During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle.

scholarly journals The role of Leishmania GP63 in the modulation of innate inflammatory response to Leishmania major infection

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0262158
Author(s):  
Aretha Chan ◽  
Jose-Mauricio Ayala ◽  
Fernando Alvarez ◽  
Ciriaco Piccirillo ◽  
George Dong ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Leishmania Major ◽  
Critical Role ◽  
Protozoan Parasite ◽  
Inflammatory Cells ◽  
Host Cells ◽  
Cell Infection ◽  
Zinc Metalloprotease

Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is known to affect millions of individuals worldwide. In recent years, we have established the critical role played by Leishmania zinc-metalloprotease GP63 in the modulation of host macrophage signalling and functions, favouring its survival and progression within its host. Leishmania major lacking GP63 was reported to cause limited infection in mice, however, it is still unclear how GP63 may influence the innate inflammatory response and parasite survival in an in vivo context. Therefore, we were interested in analyzing the early innate inflammatory events upon Leishmania inoculation within mice and establish whether Leishmania GP63 influences this initial inflammatory response. Experimentally, L. major WT (L. majorWT), L. major GP63 knockout (L. majorKO), or L. major GP63 rescue (L. majorR) were intraperitoneally inoculated in mice and the inflammatory cells recruited were characterized microscopically and by flow cytometry (number and cell type), and their infection determined. Pro-inflammatory markers such as cytokines, chemokines, and extracellular vesicles (EVs, e.g. exosomes) were monitored and proteomic analysis was performed on exosome contents. Data obtained from this study suggest that Leishmania GP63 does not significantly influence the pathogen-induced inflammatory cell recruitment, but rather their activation status and effector function. Concordantly, internalization of promastigotes during early infection could be influenced by GP63 as fewer L. majorKO amastigotes were found within host cells and appear to maintain in host cells over time. Collectively this study provides a clear analysis of innate inflammatory events occurring during L. major infection and further establish the prominent role of the virulence factor GP63 to provide favourable conditions for host cell infection.

scholarly journals Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

2002 ◽  
Vol 184 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Akiko Nishikawa ◽  
Jay B. Poster ◽  
Yoshifumi Jigami ◽  
Neta Dean
Keyword(s):  
Golgi Apparatus ◽  
Single Copy ◽  
Partial Loss ◽  
Phenotypic Analysis ◽  
Nucleotide Sugar ◽  
Yeast Viability ◽  
Mammalian Homologue ◽  
Hyphal Formation ◽  
Mannan Synthesis

ABSTRACT Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.

scholarly journals Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan Parasite Leishmania major

2004 ◽  
Vol 72 (4) ◽  
pp. 1920-1928 ◽  
Author(s):  
Pascale Kropf ◽  
Marina A. Freudenberg ◽  
Manuel Modolell ◽  
Helen P. Price ◽  
Shanti Herath ◽  
...  
Keyword(s):  
Immune Response ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Parasite Growth ◽  
Host Cells ◽  
Effective Control ◽  
Intracellular Parasites ◽  
Oxide Synthase

ABSTRACT The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasite Leishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control of Leishmania infection in vivo.

scholarly journals Heterologous Expression of Trypanosoma cruzi trans-Sialidase in Leishmania major Enhances Virulence

2000 ◽  
Vol 68 (5) ◽  
pp. 2728-2734 ◽  
Author(s):  
M. Belen Carrillo ◽  
Wenda Gao ◽  
Macario Herrera ◽  
Joseph Alroy ◽  
Jeffrey B. Moore ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Heterologous Expression ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Macrophage Infection ◽  
Cutaneous Lesions ◽  
New Approach ◽  
Extracellular Milieu ◽  
Expression Construct

ABSTRACT Earlier studies showed that mice primed for a few hours with thetrans-sialidase (TS) of Trypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection. These studies suggest that TS affects parasite virulence independent of antigenic stimulation. Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite. We tested this hypothesis by expressing heterologous TS in Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product α2-3-linked sialic acid. Leishmania cells transfected with a T. cruzi TS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu. TS expression did not affect L. majorbinding to and entry into cultured macrophages or its tropism for macrophage infection in vivo. However, TS-expressing L. major exhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro- and microscopic examination of cutaneous lesions. Several genetic tests proved that the enhanced virulence was directly attributable to TS expression. The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage on T. cruzi and transgenic L. major. These data suggest that heterologous expression of T. cruzi virulence factors inLeishmania may provide a new approach for dissecting their function in vivo.

scholarly journals Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1159
Author(s):  
Vanessa Adaui ◽  
Constanze Kröber-Boncardo ◽  
Christine Brinker ◽  
Henner Zirpel ◽  
Julie Sellau ◽  
...  
Keyword(s):  
New World ◽  
Reverse Genetics ◽  
Leishmania Major ◽  
Genetic Manipulation ◽  
Protozoan Parasite ◽  
Single Copy ◽  
Leishmania Braziliensis ◽  
Old World ◽  
Tegumentary Leishmaniasis ◽  
Null Mutants

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

Site of Lysosome Production During Hepatic Injury

1969 ◽  
Vol 27 ◽  
pp. 348-349
Author(s):  
Nalin J. Unakar
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mouse Liver ◽  
Hepatic Injury ◽  
Close Approximation ◽  
Experimental Conditions ◽  
Toxic Injury

The increased number of lysosomes as well as the close approximation of lysosomes to the Golgi apparatus in tissue under variety of experimental conditions is commonly observed. These observations suggest Golgi involvement in lysosomal production. The role of the Golgi apparatus in the production of lysosomes in mouse liver was studied by electron microscopy of liver following toxic injury by CCI4.

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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