Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms

108

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 ␤1-2 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 lesionderived 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.Leishmania species are sandfly-transmitted protozoan parasites that cause a number of human diseases, ranging from self-healing cutaneous lesions to fatal visceral infections, afflicting more than 12 million people worldwide (www.who.int/ inf-fs/en/fact116.html). These parasites develop within the midgut of the sandfly vector, initially as rapidly dividing procyclic promastigotes and subsequently as non-dividing, but highly virulent, metacyclic promastigotes. Upon transmission to the mammalian host, metacyclic promastigotes invade macrophages and differentiate to the amastigote stage that proliferates within the phagolysosomal compartment. Parasite survival within the highly acidic and hydrolytic environment of the phagolysosome is likely to involve the induction of a number of biochemical processes, such as the activation of a heat shock response (1) and the stage-specific expression of specific nutrient transporters (2).It has recently been reported that one or more mannose (Man)-containing molecules are essential for L. mexicana survival in macrophages and infectivity in animals (3, 4). Specifically, targeted deletion of genes involved in GDP-Man synthesis, such as phosphomannomutase and GDP-Man pyrophosphorylase (GDP-MP), 1 did not affect parasite growth in rich culture medium but completely attenuated infectivity in macrophages and in highly susceptible BALB/c mice (3). The GDP-Man-negative mutant, GDPMP, was shown to be deficient in a range of cell surface and secreted mannose-containing molecules, including several abunda...

Section: Methodsmentioning

confidence: 99%

Evidence That Intracellular β1-2 Mannan Is a Virulence Factor in Leishmania Parasites

Ralton¹,

Naderer²,

Piraino

et al. 2003

108

“…In vitro culture of amastigote-like forms (axenic amastigotes) involved subpassage at 32°C in Schneider's Drosophila medium (Life Technologies, Inc.) at pH 5.5, containing 20% (v/v) fetal bovine serum, in the presence of 5% (v/v) CO 2 (32). The L. mexicana ⌬CPB null mutant was grown in the presence of 50 g ml Ϫ1 hygromycin B (Sigma) and 10 g ml Ϫ1 phleomycin (Cayla, France) (27).…”

Section: Methodsmentioning

confidence: 99%

The Stage-regulated Expression of Leishmania mexicanaCPB Cysteine Proteases Is Mediated by an Intercistronic Sequence Element

Brooks

Denise

Westrop³

et al. 2001

The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3-untranslated region and intercistronic sequence of CPB2.8 showed upregulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclicspecific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.

“…Metacyclic promastigotes of L. major were purified from a stationary phase population of cells using the agglutination assay described by Sacks et al (25). Amastigotes of L. mexicana were purified from infected BALB/c mice or grown axenically as described (26). The bloodstream form of Trypanosoma brucei strain 427 was grown in rats and isolated by DEAE ionexchange chromatography as described (27).…”

Section: Cultivation Of L Major and L Mexicana-promastigotes Of L mentioning

confidence: 99%

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

Williams

Kelly

Mottram

et al. 2003

Cytosolic 3-mercaptopyruvate sulfurtransferases (EC 2.8.1.2) of Leishmania major and Leishmania mexicana have been cloned, expressed as active enzymes in Escherichia coli, and characterized. The leishmanial singlecopy genes predict a sulfurtransferase that is structurally peculiar in possessing a C-terminal domain of some 70 amino acids. Homologous genes of Trypanosoma cruzi and Trypanosoma brucei encode enzymes with a similar C-terminal domain, suggesting that this feature, not known in any other sulfurtransferase, is a characteristic of trypanosomatid parasites. Short truncations of the C-terminal domain resulted in misfolded inactive proteins, demonstrating that the domain plays some key role in facilitating correct folding of the enzymes. The leishmanial recombinant enzymes exhibited high activity toward 3-mercaptopyruvate and catalyzed the transfer of sulfane sulfur to cyanide to form thiocyanate. They also used thiosulfate as a substrate and reduced thioredoxin as the accepting nucleophile, the latter being oxidized. The enzymes were expressed in all life cycle stages, and the expression level was increased under peroxide or hypo-sulfur stress. The results are consistent with the enzymes having an involvement in the synthesis of sulfur amino acids per se or iron-sulfur centers of proteins and the parasite's management of oxidative stress.