Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

Abstract

A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.

Keywords: Leishmania; central carbon metabolism; macrophages; reductive stress; virulence.

Figure 1.. Leishmania replicate within the mature phagolysosome compartment of macrophages.

This compartment is predicted to contain a range of carbon sources (sugars, amino acids, and fatty acids) and essential nutrients (major auxotrophic requirements listed in insert) that are delivered to the phagolysosome via different endocytic pathways, autophagy, lysosomal membrane transporters, and fusion with the endoplasmic reticulum (ER). Macromolecules delivered to this compartment are degraded by a barrage of luminal hydrolases or internalized by amastigotes and degraded within their own hydrolytically active lysosomes, or both. Arg, arginine; EE, early endosome; Glc, glucose; Glc6P, glucose 6-phosphate; GlcA, glucuronic acid; GlcN, glucosamine; His, histidine; Ile, isoleucine; LE, late endosome; Leu, leucine; Lys, lysine; Man, mannose; Phe, phenylalanine; Rib, ribose; TAG, triacylglycerol; Trp, tryptophan; Tyr, tyrosine; Val, valine; Xyl, xylose.

Figure 2.. Intracellular amastigotes exhibit a stringent metabolic response.

The differentiation of Leishmania promastigotes (insect stage) to amastigotes (macrophage host) is associated with major changes in central carbon metabolism. Promastigotes exhibit high rates of glucose and (non-essential) amino acid uptake that are co-catabolized via the major pathways of central metabolism. Promastigotes also take up fatty acids, but these are primarily incorporated into membrane lipids and not used as carbon sources (downward arrow). Amastigotes also preferentially use glucose as a carbon source. However, they exhibit much lower (~10-fold) rates of sugar and amino acid uptake and overflow metabolism (note that amastigotes continue to take up essential amino acids but primarily use these for protein synthesis). Amastigotes also actively catabolize fatty acids in the tricarboxylic acid (TCA) cycle, as a result of reduced glucose uptake. The downregulation of hexose/amino acid uptake in amastigotes (stringent response) is hardwired to differentiation, as it occurs in vitro irrespective of nutrient levels and is coupled to a reduced growth rate .

Figure 3.. Carbon metabolism of Leishmania amastigotes.

Leishmania amastigotes appear to depend primarily on the uptake and catabolism of sugars scavenged from the macrophage phagolysosome. Hexose phosphates are catabolized in the glycolytic and pentose phosphate pathway (PPP) and converted to intracellular and surface glycoconjugates (GPI, N-glycans, mannogen). Key enzymes involved in glycolysis are partially or exclusively sequestered within glycosomes (modified peroxisomes), and ATP and NAD ⁺ within this organelle are regenerated by fermentation of phosphoenolpyruvate to succinate (succinate fermentation pathway, or SFP) or pyruvate . The end-products of glycosomal catabolism are further catabolized in the mitochondrion, together with acetyl-CoA generated by fatty acid β-oxidation, to produce anabolic precursors, such as glutamate. Most of the glutamate (and other non-essential amino acids) in amastigotes is synthesized de novo rather than taken up from macrophages. Excess NADH production in the mitochondrion might lead to increased endogenous reactive oxygen species (ROS) production via the respiratory chain. The gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBP), is also required for amastigote survival in vivo. This enzyme is sequestered in glycosomes with phosphofructokinase (PFK) and might allow amastigotes to transiently use other carbon sources or regulate glycolytic fluxes by cycling FBP back to fructose 6-phosphate (futile cycling), or both. αKG, α-ketoglutarate; AcCoA, acetyl-CoA; Fru6P, fructose-6-phosphate; Glc6P, glucose-6-phosphate; GlcNAc6P, N-acetylglucosamine-6-phosphate; Glu, glutamate; Man6P, mannose-6-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Rib5P, ribose-5-phosphate; Triose-P, triose phosphates.