Metabolic Plasticity Aids Amphotropism of Coxiella burnetii

Abstract

Coxiella burnetii, the causative agent of query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms (e.g., mammals and arthropods) and cell types. This amphotropic nature of C. burnetii, in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, CbΔpckA, unable to undergo gluconeogenesis. Both the parental strain and CbΔpckA mutant exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, interstitial fluid-modeled medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by the CbΔpckA mutant in specific cell lines. The CbΔpckA mutant was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen.

Keywords: Coxiella burnetii; amphotropism; gluconeogenesis; glycolysis; interstitial fluid; intracellular parasites; metabolic plasticity; obligate; physiological medium; virulence.

FIG 1

C. burnetii intracellular replication potential is dependent on host cell type and nutritional environment. C. burnetii intracellular replication was measured during infection of J774A.1 (A), JEG-3 (B), THP-1 (C), and VERO (D) cells cultured in complete RPMI or IFmM. Replication was determined via quantification of GE every 2 days for 8 days. Depicted data illustrate means ± SEM (n = 3 to 6). *, P < 0.05; **, P < 0.01 (unpaired Student's t test).

FIG 2

Medium glucose and amino acid levels directly influence C. burnetii intracellular replication in a cell type-dependent manner. To assess the influence of specific nutrients provided to the host on C. burnetii intracellular replication, J774A.1 (A), VERO (B), and JEG-3 (C) cells were infected with C. burnetii expressing GFP and cultured in modified IFmM with increasing amounts of glucose or altered concentrations of amino acids (AAs). C. burnetii yields were determined via enumeration of GE at 5 days p.i., and values presented as percent of control (complete IFmM, 100%, dashed line). Depicted data illustrate means ± SEM (n = 3 to 8). *, P < 0.05; ****, P < 0.0001 (one-way analysis of variance [ANOVA] with Dunnett’s posttest applied to panels A and C). (D) Representative fluorescence micrographs of GFP expressing C. burnetii 5 days p.i. under the conditions described for panels A to C.

FIG 3

Intracellular pools of glutamate and glucose are host cell type specific. Intracellular concentrations of glutamate (A) and glucose (B) in J774A.1, JEG-3, and VERO cells maintained in RPMI or IFmM were quantified. Data are presented as mean intracellular concentration per 10⁷ cells as determined via direct cell counts. Depicted data illustrate the means ± SEM (n = 3 to 7). P < 0.05 (one-way ANOVA with Tukey’s posttest).

FIG 4

Gluconeogenic capacity is necessary for C. burnetii amphotropic fitness. Intracellular growth of the Pt, CbΔpckA, and CbΔpckA::pckA strains were compared during infection of J774A.1 (A), JEG-3 (B), THP-1 (C), and VERO (D) cells cultured in IFmM (blue) or RPMI (gray). Replication was determined via quantification of GE every 2 days for 8 days. Depicted data illustrate means ± SEM (n = 3 to 6). Asterisks in blue and gray indicate differences between the Pt and CbΔpckA strains during infection of host cells cultured in IFmM or RPMI, respectively. *, P < 0.05; **, P < 0.01 (unpaired Student's t test). Brackets indicate the change in final (i.e., day 8) yields between the Pt and CbΔpckA strains.

FIG 5

Metabolic plasticity enhances C. burnetii virulence. (A) D. melanogaster survival was assessed daily for 40 days following mock challenge or infection with the Pt, CbΔpckA, and CbΔpckA::pckA strains. Depicted data illustrate a single experiment (n = 50 flies) and is representative of 2 independent experiments. *, P < 0.05; ****, P < 0.0001 (Mantel-Cox compared to control). The ability of the CbΔpckA strain to infect D. melanogaster S2 (B), Ixodes scapularis (ISE6) (C), and Dermacentor andersoni (DAE100) (D) cells was compared to the Pt strain every 2 days for 10 days. Depicted data illustrate means ± SEM (n = 3 to 4). *, P < 0.05; **, P < 0.01 (unpaired Student's t test). Quantification of intracellular concentrations of glucose (E) and glutamate (F) in homogenates of S2, DAE100, and ISE6 cells. Depicted data illustrate means ± SEM (n = 3 to 5). *, P < 0.05 (one-way ANOVA with Tukey’s posttest).