Defining the Metabolic Pathways and Host-Derived Carbon Substrates Required for Francisella tularensis Intracellular Growth

Abstract

Francisella tularensis is a Gram-negative, facultative, intracellular bacterial pathogen and one of the most virulent organisms known. A hallmark of F. tularensis pathogenesis is the bacterium's ability to replicate to high densities within the cytoplasm of infected cells in over 250 known host species, including humans. This demonstrates that F. tularensis is adept at modulating its metabolism to fluctuating concentrations of host-derived nutrients. The precise metabolic pathways and nutrients utilized by F. tularensis during intracellular growth, however, are poorly understood. Here, we use systematic mutational analysis to identify the carbon catabolic pathways and host-derived nutrients required for F. tularensis intracellular replication. We demonstrate that the glycolytic enzyme phosphofructokinase (PfkA), and thus glycolysis, is dispensable for F. tularensis SchuS4 virulence, and we highlight the importance of the gluconeogenic enzyme fructose 1,6-bisphosphatase (GlpX). We found that the specific gluconeogenic enzymes that function upstream of GlpX varied based on infection model, indicating that F. tularensis alters its metabolic flux according to the nutrients available within its replicative niche. Despite this flexibility, we found that glutamate dehydrogenase (GdhA) and glycerol 3-phosphate (G3P) dehydrogenase (GlpA) are essential for F. tularensis intracellular replication in all infection models tested. Finally, we demonstrate that host cell lipolysis is required for F. tularensis intracellular proliferation, suggesting that host triglyceride stores represent a primary source of glycerol during intracellular replication. Altogether, the data presented here reveal common nutritional requirements for a bacterium that exhibits characteristic metabolic flexibility during infection.IMPORTANCE The widespread onset of antibiotic resistance prioritizes the need for novel antimicrobial strategies to prevent the spread of disease. With its low infectious dose, broad host range, and high rate of mortality, F. tularensis poses a severe risk to public health and is considered a potential agent for bioterrorism. F. tularensis reaches extreme densities within the host cell cytosol, often replicating 1,000-fold in a single cell within 24 hours. This remarkable rate of growth demonstrates that F. tularensis is adept at harvesting and utilizing host cell nutrients. However, like most intracellular pathogens, the types of nutrients utilized by F. tularensis and how they are acquired is not fully understood. Identifying the essential pathways for F. tularensis replication may reveal new therapeutic strategies for targeting this highly infectious pathogen and may provide insight for improved targeting of intracellular pathogens in general.

Keywords: Francisella tularensis; GdhA; GlpA; carbon metabolism; intracellular pathogen.

FIG 1

An overview of Francisella tularensis subsp. tularensis Schu S4 central carbon metabolism. Labeled enzymes (red) are predicted to be required for the flux of specific carbon substrates (blue) for F. tularensis central carbon metabolism. Orange arrows indicate reactions that are specific to gluconeogenesis. PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; acetyl-CoA, acetyl coenzyme A; PfkA, phosphofructokinase (FTT_0801); GlpX, fructose 1,6-bisphosphatase (FTT_1631); GlpK, glycerol kinase (FTT_0130); GlpA, glycerol 3-phosphate dehydrogenase (FTT_0132); PckA, phosphoenolpyruvate carboxykinase (FTT_0449); PpdK, pyruvate phosphate dikinase (FTT_0250); MaeA, malic enzyme (FTT_0917); GdhA, glutamate dehydrogenase (FTT_0380).

FIG 2

F. tularensis GlpX is essential for replication on gluconeogenic carbon substrates, within host macrophages, and in a murine model of infection. (A) Terminal OD₆₀₀ of WT Schu S4, ΔpfkA, and ΔglpX strains after 48 h of growth in CDM and CDM supplemented with glucose or glutamate at a final concentration of 0.4%. (B) Intracellular replication of WT Schu S4, ΔpfkA, ΔglpX, and ΔglpX pglpX strains in BMDMs, as indicated via relative light units (RLU) measured every 15 min over a 36-hour period. (C) Growth of WT Schu S4 and (D) ΔglpX strains in BMDMs cultured with or without 150 µM AICAR and/or glucose at a concentration of 4.5 g/liter. Growth was measured via luminescence read every 15 min over 36 h. Each growth curve represents one of three independent experiments, and each data point represents the average of three technical triplicates. (E) Organ burdens of mice 3 days post intranasal inoculation with WT Schu S4, ΔpfkA, and ΔglpX strains. Data are pooled from three independent experiments. **, P < 0.01, and ***, P < 0.001, as determined by Student's t test.

FIG 3

Growth of ΔpckA and ΔppdK mutants in defined medium, host cells, and a murine model of infection. Terminal OD₆₀₀ of (A) ΔpckA and (B) ΔppdK and ΔppdK pppdK strains after 48 h of growth in CDM and CDM supplemented with glucose or glutamate at a final concentration of 0.4%. Data are pooled from three triplicate wells from three independent experiments (mean ± standard deviation [SD]). (C) The intracellular growth kinetics of ΔpckA, ΔppdK, and ΔppdK ΔpckA mutants cultured in high-glucose (4.5 g/liter) DMEM, as indicated via RLU measured every 15 min over a 36-hour period. The data shown represent three independent experiments, and each data point represents the average of three technical replicates. (D) Organ burdens of mice 3 days post intranasal inoculation with WT Schu S4, ΔppdK, ΔpckA, or ΔppdK ΔpckA mutants. Data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, as determined by Student's t test.

FIG 4

GdhA fuels gluconeogenesis by shuttling carbon into the TCA cycle. (A) Terminal OD₆₀₀ of ΔgdhA cells after 48 h of growth in CDM and CDM supplemented with glucose or glutamate at a final concentration of 0.4%. Data are pooled from three triplicate wells from three independent experiments (mean ± SD). (B) The intracellular growth kinetics of WT Schu S4, ΔgdhA, and ΔgdhA pgdhA strains within BMDMs, as indicated via RLU measured every 15 min over a 36-h period. (C) ΔgdhA strains expressing the luciferase (Lux) reporter of intracellular growth in BMDMs cultured with or without 150 µM AICAR and/or glucose at a concentration of 4.5 g/liter. Each growth curve represents one of three independent experiments, and each data point represents the average of three technical triplicates. (D) Organ burdens of mice 3 days post intranasal inoculation with WT Schu S4 or the ΔgdhA mutant. Data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, as determined by Student's t test.

FIG 5

Glycerol metabolism is essential for F. tularensis intracellular replication. (A) Terminal OD₆₀₀ of WT Schu S4, the glpKA insertion mutant, and the corresponding pglpKAF complemented strain grown in CDM and CDM supplemented with glucose, glycerol, or G3P after 48 h of growth. Data are pooled from three triplicate wells from three independent experiments (mean ± SD). (B) Growth curves of WT Schu S4, the glpKA mutant, and the glpKAF complemented strain harboring the LUX reporter within BMDMs. Intracellular bacterial growth was measured via luminescence (RLU), read every 15 min over a 36-hour period. (C) Growth of ΔglpA mutant expressing the LUX reporter of intracellular growth in BMDMs cultured with or without 150 µM AICAR and/or glucose at a concentration of 4.5 g/liter. Each growth curve represents one of three independent experiments, and each data point represents the average of three technical triplicates. (D) Organ burdens of mice 3 days post intranasal inoculation with WT Schu S4 and the glpKA insertional mutant. Data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, as determined by Student's t test.

FIG 6

Active host cell lipolysis is required for efficient F. tularensis intracellular replication. (A) Growth curve of WT Schu S4 cells harboring a LUX reporter within BMDMs cultured in high-glucose (4.5 g/liter) DMEM with or without Atglistatin at indicated concentrations. Intracellular bacterial growth was measured via luminescence (RLU), read every 15 min over a 24-h period. Data represent the mean pooled from 3 replicates in 3 independent experiments. (B) Fold change in WT Schu S4 burden between 24 and 4 h postinfection of WT and ATGL knockdown BMDMs. Data are pooled from three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, as determined by Student's t test).