Dual Isotopologue Profiling of Bacterial Pathogens and their Host Cells: Metabolic Adaptation of Human Macrophages and Fallopian Tube Cells to Intracellular Chlamydia trachomatis

Chlamydia trachomatis is a Gram-negative bacterium that utilizes multiple host-derived substrates to ensure its intracellular survival. In this study, human fallopian tube (HFT) cells, and human macrophages polarized toward a pro-inflammatory (M1-like) or anti-inflammatory (M2-like) state were infected with C. trachomatis and cocultivated in the presence of [U-¹³C₆]glucose. Samples were analyzed in toto by dual isotopologue profiling with a focus on specific bacterial and host-specific metabolites. Immunofluorescence and ultrastructural analysis, as well as detection of the bacteria-specific metabolites (i.e., the branched-chain iso-C15:0 and anteiso-C15:0 fatty acids, and the cell wall component meso-diaminopimelic acid), confirmed that HFT cells and M2-like, but not M1-like macrophages, allow replication of C. trachomatis. The ¹³C-labeling patterns in these metabolites reflected their known biosynthetic pathways, but also upstream carbon fluxes via the uptake of host amino acids and glucose phosphate into the intracellular bacteria. Differential analysis of infected vs noninfected host cells showed that, in HFT cells and M2-like macrophages, the chlamydial infection upregulated glucose uptake into the host cells, glucose conversion into pyruvate and lactate via host glycolysis, and release of lactate into the medium. The rates of these processes were higher in HFT cells than in M2-like macrophages. We here establish dual isotopologue profiling as a suitable method to analyze the dynamics of host-intracellular pathogen interactions.