<i>Orientia tsutsugamushi</i> infection reduces host gluconeogenic but not glycolytic substrates

<i>Orientia tsutsugamushi</i> a causal agent of scrub typhus, is an obligate intracellular bacterium that, akin to other rickettsiae, is dependent on host cell-derived nutrients for survival and thus pathogenesis. Based on limited experimental evidence and genome-based <i>in silico</i> predictions, <i>O. tsutsugamushi</i> is hypothesized to parasitize host central carbon metabolism (CCM). Here, we (re-)evaluated <i>O. tsutsugamushi</i> dependency on host cell CCM as initiated by glucose and glutamine. <i>Orientia</i> infection had no effect on host glucose and glutamine consumption or lactate accumulation, indicating no change in overall flux through CCM. However, host cell mitochondrial activity and ATP levels were reduced during infection and correspond with lower intracellular glutamine and glutamate pools. To further probe the essentiality of host CCM in <i>O. tsutsugamushi</i> proliferation, we developed a minimal medium for host cell cultivation and paired it with chemical inhibitors to restrict the intermediates and processes related to glucose and glutamine metabolism. These conditions failed to negatively impact <i>O. tsutsugamushi</i> intracellular growth, suggesting the bacterium is adept at scavenging from host CCM. Accordingly, untargeted metabolomics was utilized to evaluate minor changes in host CCM metabolic intermediates across <i>O. tsutsugamushi</i> infection and revealed that pathogen proliferation corresponds with reductions in critical CCM building blocks, including amino acids and TCA cycle intermediates, as well as increases in lipid catabolism. This study directly correlates <i>O. tsutsugamushi</i> proliferation to alterations in host CCM and identifies metabolic intermediates that are likely critical for pathogen fitness.IMPORTANCEObligate intracellular bacterial pathogens have evolved strategies to reside and proliferate within the eukaryotic intracellular environment. At the crux of this parasitism is the balance between host and pathogen metabolic requirements. The physiological basis driving <i>O. tsutsugamushi</i> dependency on its mammalian host remains undefined. By evaluating alterations in host metabolism during <i>O. tsutsugamushi</i> proliferation, we discovered that bacterial growth is independent of the host's nutritional environment but appears dependent on host gluconeogenic substrates, including amino acids. Given that <i>O. tsutsugamushi</i> replication is essential for its virulence, this study provides experimental evidence for the first time in the post-genomic era of metabolic intermediates potentially parasitized by a scrub typhus agent.