Jacob R. Gillingham

The Gram-positive pathogen Streptococcus pneumoniae, like the majority of bacteria, contains a peptidoglycan-based cell wall whose structure is highly dependent on the action of penicillin-binding proteins (PBPs). While the β-lactam antibiotics have been employed as an antimicrobial strategy for nearly a century, much remains unclear about how inhibitor structure informs potency and PBP isoform selectivity. Here, we obtained high-resolution structures (S. pneumoniae PBP1b cocrystallized with 6 β-lactams. Surprisingly, 2 structures feature a noncanonical conformation of the covalent "acyl-enzyme complex." To clarify how protein-ligand interactions mediate inhibitor binding, we applied molecular modeling and molecular mechanics-based dynamics analyses. Our analyses illustrate how seemingly minimal changes to inhibitor structure modulate β-lactam binding mode and inhibitor potency, as described by the metric k_inact/K_I. Furthermore, we demonstrate that persistent interaction in the covalent acyl-enzyme complex between the inhibitor carboxylate and a highly conserved three-residue motif is not fully predictive of k_inact/K_I for PBP1b. In silico modeling suggests that the noncovalent preacyl complex may leverage this interaction, but a postacylation change in ligand conformation may accompany acylation in some inhibitors. The elucidation of key PBP1b ligand-receptor interactions pre- and postacylation will inform the rational design of novel PBP inhibitors and probes.

The progression of metabolic dysfunction–associated steatotic liver disease (MASLD) to metabolic dysfunction–associated steatohepatitis (MASH) involves alterations in both liver-autonomous and systemic metabolism that influence the liver’s balance of fat accretion and disposal. Here, we quantify the contributions of hepatic oxidative pathways to liver injury in MASLD-MASH. Using NMR spectroscopy, UHPLC-MS, and GC-MS, we performed stable isotope tracing and formal flux modeling to quantify hepatic oxidative fluxes in humans across the spectrum of MASLD-MASH, and in mouse models of impaired ketogenesis. In humans with MASH, liver injury correlated positively with ketogenesis and total fat oxidation, but not with turnover of the tricarboxylic acid cycle. Loss-of-function mouse models demonstrated that disruption of mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting step of ketogenesis, impairs overall hepatic fat oxidation and induces an MASLD-MASH–like phenotype. Disruption of mitochondrial β-hydroxybutyrate dehydrogenase (BDH1), the terminal step of ketogenesis, also impaired fat oxidation, but surprisingly did not exacerbate steatotic liver injury. Taken together, these findings suggest that quantifiable variations in overall hepatic fat oxidation may not be a primary determinant of MASLD-to-MASH progression, but rather that maintenance of ketogenesis could serve a protective role through additional mechanisms that extend beyond overall rates of fat oxidation.

Ketogenesis is a dynamic metabolic conduit supporting hepatic fat oxidation particularly when carbohydrates are in short supply. Ketone bodies may be recycled into anabolic substrates, but a physiological role for this process has not been identified. Here, we use mass spectrometry–based ¹³ C-isotope tracing and shotgun lipidomics to establish a link between hepatic ketogenesis and lipid anabolism. Unexpectedly, mouse liver and primary hepatocytes consumed ketone bodies to support fatty acid biosynthesis via both de novo lipogenesis (DNL) and polyunsaturated fatty acid (PUFA) elongation. While an acetoacetate intermediate was not absolutely required for ketone bodies to source DNL, PUFA elongation required activation of acetoacetate by cytosolic acetoacetyl–coenzyme A synthetase (AACS). Moreover, AACS deficiency diminished free and esterified PUFAs in hepatocytes, while ketogenic insufficiency depleted PUFAs and increased liver triacylglycerols. These findings suggest that hepatic ketogenesis influences PUFA metabolism, representing a molecular mechanism through which ketone bodies could influence systemic physiology and chronic diseases.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, k_inact/K_I, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, k_inact/K_I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell k_inact/K_I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro k_inact/K_I data and show a comparable relationship to previously established IC₅₀ values. These results support the validity of our in vivo k_inact/K_I method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

: The progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) to metabolic-dysfunction-associated steatohepatitis (MASH) involves complex alterations in both liver-autonomous and systemic metabolism that influence the liver’s balance of fat accretion and disposal. Here, we quantify the relative contribution of hepatic oxidative pathways to liver injury in MASLD-MASH. Using NMR spectroscopy, UHPLC-MS, and GC-MS, we performed stable-isotope tracing and formal flux modeling to quantify hepatic oxidative fluxes in humans across the spectrum of MASLD-MASH, and in mouse models of impaired ketogenesis. We found in humans with MASH, that liver injury correlated positively with ketogenesis and total fat oxidation, but not with turnover of the tricarboxylic acid cycle. The use of loss-of-function mouse models demonstrated that disruption of mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting step of ketogenesis, impairs overall hepatic fat oxidation and induces a MASLD-MASH-like phenotype. Disruption of mitochondrial β-hydroxybutyrate dehydrogenase (BDH1), the terminal step of ketogenesis, also impaired fat oxidation, but surprisingly did not exacerbate steatotic liver injury. Taken together, these findings suggest that quantifiable variations in overall hepatic fat oxidation may not be a primary determinant of MASLD-to-MASH progression, but rather, that maintenance of hepatic ketogenesis could serve a protective role through additional mechanisms that extend beyond quantified overall rates of fat oxidation.

Summary: Therapeutic interventions targeting hepatic lipid metabolism in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) remain elusive. Using mass spectrometry-based stable isotope tracing and shotgun lipidomics, we established a novel link between ketogenesis and MASLD pathophysiology. Our findings show that mouse liver and primary hepatocytes consume ketone bodies to support fatty acid (FA) biosynthesis via both de novo lipogenesis (DNL) and FA elongation. Analysis of ¹³ C-labeled FAs in hepatocytes lacking mitochondrial D-β-hydroxybutyrate dehydrogenase (BDH1) revealed a partial reliance on mitochondrial conversion of D-βOHB to acetoacetate (AcAc) for cytoplasmic DNL contribution, whereas FA elongation from ketone bodies was fully dependent on cytosolic acetoacetyl-CoA synthetase (AACS). Ketone bodies were essential for polyunsaturated FA (PUFA) homeostasis in hepatocytes, as loss of AACS diminished both free and esterified PUFAs. Ketogenic insufficiency depleted liver PUFAs and increased triacylglycerols, mimicking human MASLD, suggesting that ketogenesis supports PUFA homeostasis, and may mitigate MASLD-MASH progression in humans.

Background: Responses of the metabolome to acute aerobic exercise may predict maximum oxygen consumption (VO ₂ max) and longer-term outcomes, including the development of diabetes and its complications. Methods: Serum samples were collected from overweight/obese trained (OWT) and normal-weight trained (NWT) runners prior to and immediately after a supervised 90-minute treadmill run at 60% VO ₂ max (NWT = 14, OWT = 11) in a cross-sectional study. We applied a liquid chromatography high-resolution–mass spectrometry–based untargeted metabolomics platform to evaluate the effect of acute aerobic exercise on the serum metabolome. Results: NWT and OWT metabolic profiles shared increased circulating acylcarnitines and free fatty acids (FFAs) with exercise, while intermediates of adenine metabolism, inosine, and hypoxanthine were strongly correlated with body fat percentage and VO ₂ max. Untargeted metabolomics-guided follow-up quantitative lipidomic analysis revealed that baseline levels of fatty acid esters of hydroxy fatty acids (FAHFAs) were generally diminished in the OWT group. FAHFAs negatively correlated with visceral fat mass and HOMA-IR. Strikingly, a 4-fold decrease in FAHFAs was provoked by acute aerobic running in NWT participants, an effect that negatively correlated with circulating IL-6; these effects were not observed in the OWT group. Machine learning models based on a preexercise metabolite profile that included FAHFAs, FFAs, and adenine intermediates predicted VO ₂ max. Conclusion: These findings in overweight human participants and healthy controls indicate that exercise-provoked changes in FAHFAs distinguish normal-weight from overweight participants and could predict VO ₂ max. These results support the notion that FAHFAs could modulate the inflammatory response, fuel utilization, and insulin resistance. Trial registration: ClinicalTrials.gov, NCT02150889. Funding: NIH DK091538, AG069781, DK098203, TR000114, UL1TR002494.