Mathematical modeling of pathogenicity of Cryptococcus neoformans

Abstract

Cryptococcus neoformans (Cn) is the most common cause of fungal meningitis worldwide. In infected patients, growth of the fungus can occur within the phagolysosome of phagocytic cells, especially in non-activated macrophages of immunocompromised subjects. Since this environment is characteristically acidic, Cn must adapt to low pH to survive and efficiently cause disease. In the present work, we designed, tested, and experimentally validated a theoretical model of the sphingolipid biochemical pathway in Cn under acidic conditions. Simulations of metabolic fluxes and enzyme deletions or downregulation led to predictions that show good agreement with experimental results generated post hoc and reconcile intuitively puzzling results. This study demonstrates how biochemical modeling can yield testable predictions and aid our understanding of fungal pathogenesis through the design and computational simulation of hypothetical experiments.

Figure 1

Model diagram of sphingolipid metabolism in Cn. Metabolites in boxes represent dependent variables that are defined through differential equations and are numbered from X₁ to X₁₉. Independent variables are numbered from X₁₀₀ to X₁₃₆. Solid arrows show flow of material. Plus signs associated with dotted arrows represent activation. The acylation state is coded as (1) C₂₆-CoA, (2) C₁₈-CoA, and (3) C₂₄-CoA; these are substrates for the DH-Cer synthase reaction or for the enzyme P-Cer synthase (see main text and Supplementary information for details). Dependent variables: Pal-CoA (X₁), palmitoyl-CoA; serine (X₂); KDHS (X₃), 3-ketodihydrosphingosine; DHS (X₄), dihydrosphingosine; dihydro-C₂₄ (X₅), dihydroceramide C₂₄; dihydro-C₂₆ (X₆), dihydroceramide C₂₆; dihydro-C₁₈ (X₇), dihydroceramide C₁₈; PHS (X₈), phytosphingosine; phyto-C₂₆ (X₉), phytoceramide C₂₆; phyto-C₂₄ (X₁₀), phytoceramide C₂₄; phyto-C₁₈ (X₁₁), phytoceramide C₁₈; Pma1 (X₁₂), newly synthesized Pma1; IPC-C₂₆ (X₁₃), inositol phosphorylceramide C₂₆; IPC-C₂₄ (X₁₄), inositol phosphorylceramide C₂₄; IPC-C₁₈ (X₁₅), inositol phosphorylceramide C₁₈; intracellular protons (X₁₆); ATP (X₁₇), adenosine-5′-triphosphate; palmitate (X₁₈); DAG (X₁₉), sn-1,2-diacylglycerol. Independent variables: palmitate ext (X₁₀₀), palmitate external; serine ext (X₁₀₁), serine external; palmitate transport (X₁₀₂); serine transport (X₁₀₃); Ac-CoA (X₁₀₄), acetyl CoA; C₂₆-CoA (X₁₀₅), very long-chain fatty acid (C₂₆-CoA); C₁₈-CoA (X₁₀₆), fatty acid (C₁₈-CoA); C₂₄-CoA (X₁₀₇), fatty acid (C₂₄-CoA); serine palmitoyltransferase (X₁₀₈); ADP, adenosine biphosphate (X₁₀₉); dihydro-CDase (X₁₁₀), dihydroceramide ceramidase; KDHS reductase (X₁₁₁), 3-ketodihydrosphingosine reductase; DH-Cer synthase (X₁₁₂), dihydroceramide synthase; phyto-CDase (X₁₁₃), phytoceramidase; hydroxylase (X₁₁₄); hydroxylase (X₁₁₅); P-Cer synthase (X₁₁₆), phytoceramide synthase; Pma1p (X₁₁₇), newly synthesized Pma1 in the ER; Sec61 (X₁₁₈), Sec61 as probable ER insertion protein; Isc1 (X₁₁₉), inositol phosphosphingolipid phospholipase C; PI (X₁₂₀), phosphatidylinositol; Ipc1 (X₁₂₁), inositol phosphorylceramide synthase; alternative respiration (X₁₂₂); NADHm (X₁₂₃), nicotinamide adenine dinucleotide; oxygen (X₁₂₄); Pma1-H⁺ATPase (X₁₂₅), synthesized plasma membrane H⁺-ATPase; H⁺ (X₁₂₆), protons external; ER–Golgi transport (X₁₂₇); H⁺ transport (X₁₂₈), proton transport; SHMT (X₁₂₉) serine hydroxymethyl transferase; Golgi membrane (X₁₃₀); Pal-CoA synthase (X₁₃₁), palmitoyl-CoA synthase; ATP total (X₁₃₂); AMP (X₁₃₃), adenosine monophosphate; Golgi–ER transport (X₁₃₄); F₀F₁-ATPase (X₁₃₅), F₀F₁-ATP synthase; H⁺ m (X₁₃₆), mitochondrial protons.

Figure 2

Simulation result of a 95% decrease in Isc1 (X₁₁₉) activity. Phyto-C₂₆ (X₉), Pma1 (X₁₂), and ATP (X₁₇) decrease. The remaining metabolites stay close to their initial values, except for IPC-C₂₆ (X₁₃), which increases. The intracellular pH decreases from 6.5 to 3.6.

Figure 3

Simulation result of an 85% decrease in Ipc1 (X₁₂₁) activity. Phyto-C₁₈ (X₁₁), IPC-C₂₆ (X₁₃), and Pma1 (X₁₂) decrease, whereas phyto-C₂₆ (X₉) and phyto-C₂₄ (X₁₀) increase. The remaining metabolites stay close to their initial values. The intracellular pH decreases from 6.5 to 5.4.

Figure 4

Measurement of intracellular ATP in Δisc1 mutant and control strains during growth at neutral and acidic pH. (A) Production of ATP is not impaired in the Δisc1 strain when exposed to a neutral pH environment. (B) Production of ATP is significantly impaired in the Δisc1 strain when exposed to a low-pH environment. Values are reported as pmol/μg protein. Results are means±s.d. of three separate experiments. ^*P<0.05, Δisc1 versus WT.