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

Abstract

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...

Properties of GDP-mannose Pyrophosphorylase, a Critical Enzyme and Drug Target in Leishmania mexicana

“…Also lost was the newly discovered ␤1-2 mannan, considered important in protection from stress. It has been suggested that the failure of the GDP-MP mutants to accumulate ␤1-2 mannan may account to a large extent for their lack of survival in vivo (22). The ⌬GDP-MP parasites were viable in cell-free culture but were unable to survive in macrophages or mice, making it a very attractive target for anti-Leishmania drug design.…”

Leishmania β-1,2-mannan is assembled on a mannose-cyclic phosphate primer

“…Leishmania lack classic carbohydrate reserves, such as glycogen or starch, but synthesize a unique class of oligosaccharides composed of linear chains of ␤-1,2-linked mannose (Man) (5)(6)(7)(8). Leishmania ␤-1,2-mannan clearly functions as a dynamic reserve material and accumulates to millimolar concentrations in infective promastigote and intracellular amastigote stages (8).…”

“…Leishmania ␤-1,2-mannan clearly functions as a dynamic reserve material and accumulates to millimolar concentrations in infective promastigote and intracellular amastigote stages (8). Mannan also accumulates in parasites subjected to physiological stresses encountered in the macrophage (8), suggesting that it might be important for intracellular survival.…”

“…Leishmania ␤-1,2-mannan clearly functions as a dynamic reserve material and accumulates to millimolar concentrations in infective promastigote and intracellular amastigote stages (8). Mannan also accumulates in parasites subjected to physiological stresses encountered in the macrophage (8), suggesting that it might be important for intracellular survival. Consistent with this notion, loss of expression of all Man-containing molecules in Leishmania mexicana by targeted deletion of the genes involved in GDP-Man biosynthesis resulted in complete loss of virulence in macrophages and susceptible mice (4,8).…”

“…Mannan also accumulates in parasites subjected to physiological stresses encountered in the macrophage (8), suggesting that it might be important for intracellular survival. Consistent with this notion, loss of expression of all Man-containing molecules in Leishmania mexicana by targeted deletion of the genes involved in GDP-Man biosynthesis resulted in complete loss of virulence in macrophages and susceptible mice (4,8). In contrast, L. mexicana mutants that had defects in assembly of some or all of their Man-rich surface glycocalyx but that retained the capacity to synthesize mannan remained virulent in macrophages (9,10).…”