Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase

Abstract

Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macrophages. To define the biochemical composition of the phagosome and carbon source requirements of intracellular stages of L. major, we investigated the role and requirement for the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP). L. major FBP was constitutively expressed in both extracellular and intracellular stages and was primarily targeted to glycosomes, modified peroxisomes that also contain glycolytic enzymes. A L. major FBP-null mutant was unable to grow in the absence of hexose, and suspension in glycerol-containing medium resulted in rapid depletion of internal carbohydrate reserves. L. major ⌬fbp promastigotes were internalized by macrophages and differentiated into amastigotes but were unable to replicate in the macrophage phagolysosome. Similarly, the mutant persisted in mice but failed to generate normal lesions. The data suggest that Leishmania amastigotes reside in a glucose-poor phagosome and depend heavily on nonglucose carbon sources. Feeding experiments with [ 13 C]fatty acids showed that fatty acids are poor gluconeogenic substrates, indicating that amino acids are the major carbon source in vivo. The need for amino acids may have forced Leishmania spp. to adapt to life in the mature phagolysosome.gluconeogenesis ͉ intracellular pathogens ͉ phagolysosome ͉ carbohydrate ͉ mannan L eishmania are parasitic protozoa that cause a spectrum of important diseases in Ͼ12 million people worldwide, ranging from self-healing cutaneous lesions to nonhealing mucocutaneous and visceral disease. There are no effective vaccines against leishmaniasis, and current drug treatments are characterized by low efficacy, high toxicity, and expense or, in the case of front-line antimonial drugs, widespread resistance (1). Infection of humans and other animal hosts is initiated by flagellated promastigotes that develop within the digestive tract of the sandfly vector and are injected into the skin during a sandfly blood meal. Promastigotes are internalized into mature phagolysosomes of a number of phagocytic host cells, including neutrophils, dendritic cells, and macrophages but proliferate only within the latter (2). The proliferative intracellular stages are aflagellate amastigotes that remain within the phagolysosome and perpetuate infection in the host through repeated cycles of macrophage infection.Very few other microbial pathogens are capable of proliferating within macrophage phagolysosomes (3), and little is known about the nutrient composition of this compartment or the metabolic responses of persistent Leishmania amastigote stages (4). Glucose uptake appears to be essential in promastigote infection of macrophages, because promastigotes of a Leishmania mexicana mutant lacking all glucose transporters are unable to infect macrophages (5). However, this mutant fails to differentiate into amastigotes in vitro (5), and the hexose requirements of this stage are largely unknown. Glucose and other hexoses are a pot...

Down-regulation of dendritic cell signaling pathways by Leishmania amazonensis amastigotes

“…While the reduced p-STAT2 level in La-infected DCs could reflect the degradation of total STAT2 proteins by the parasites, the mechanisms by which La reduces phosphorylation of STAT1 are not known. Although various types of Leishmania-derived phosphatases, such as LPTP1, bisphosphatase and secreted acid phosphatase, have recently been identified (Naderer et al, 2006;Nascimento et al, 2006), our studies with phosphatase inhibitor (bpV)-pretreated parasites suggested a limited role for parasite-derived phosphatases in the dephosphorylation of STAT1 (Fig.5D). Whether La infection triggers the activation of a host cellular phosphatase(s) awaits further investigation.…”

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…Li ϩ -sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulindependent diabetes (5,6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7,8).…”

Metabolic Changes in Glucose Transporter-deficient Leishmania mexicana and Parasite Virulence

“…The ability of these glucogenic precursors to promote synthesis of polysaccharides argues that a bona fide gluconeogenic pathway exists in Leishmania parasites. Furthermore, a recent study (40) reveals that this pathway is essential for the survival of amastigotes of L. major, because a null mutant in the gene encoding the gluconeogenic enzyme FBPase is not viable in the amastigote stage of the life cycle.…”

“…A recent publication by Naderer et al (40) has demonstrated that a null mutant (⌬fbp) in the gene encoding the gluconeogenic enzyme FBPase in L. major is also unable to survive as amastigotes, and in this regard, has a phenotype quite similar to that of the glucose transporter null mutant. Together, these results underscore the importance of carbohydrates in amastigotes and imply that the intracellular stage of the life cycle requires both glucose transporters and gluconeogenesis for viability.…”