Johnson, R. J. | Search bioRxiv

bioRxiv2026

The cellular esterase FrmB controls metabolic homeostasis and small colony variant formation in Staphylococcus aureus

Staphylococcus aureus is a gram-positive bacterium that commonly colonizes the nasal passage, axilla, groin, and gastrointestinal tract of adults and adolescents. Although frequently nonpathogenic, S. aureus can infect most tissue types with clinical outcomes ranging from mild to fatal. The ability of S. aureus to persist and infect multiple body sites is driven largely by its metabolism. S. aureus can metabolize a wide range of niche-specific carbon sources, which contributes significantly to its persistence. Specifically, the utilization of glycolytic intermediates and pyruvate are associated with S. aureus bacterial burden and survival in the host. In this study, we establish the biological role of a serine hydrolase, FrmB, and evaluate its impact on S. aureus carbon metabolism. Using targeted metabolomics on S. aureus strains with and without FrmB, we find that FrmB is required for central carbon metabolic homeostasis. Mechanistically, we find that FrmB controls the enzymatic activity of the crucial rate-limiting enzyme, pyruvate dehydrogenase, which links glycolysis to the downstream tricarboxylic acid (TCA) cycle. Importantly, we find that the metabolic derangements in the absence of FrmB impact the ability of S. aureus to utilize pyruvate as a primary carbon source and reduce fitness. Finally, we demonstrate that FrmB is important for the formation of small colony variants (SCVs), a clinically relevant metabolic transition that is associated with chronic infection and antibiotic resistance.

microbiology↗

bioRxiv2024

Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH)

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance. SignificanceRising antimalarial drug resistance threatens current gains in malaria control. New antimalarial drugs are urgently needed. Unfortunately, many drug candidates have poor drug-like properties, such as poor absorbability in the gastrointestinal tract, or poor accumulation at the site of action. This can be overcome by prodrugging, the addition of prodrug groups which mask poor drug features until they are removed by an activating enzyme. Here, we show that a red blood cell enzyme, acylpeptide hydrolase, is taken up by malaria parasites and serves as the activating enzyme for multiple lipophilic ester prodrugs. Our findings highlight a novel mechanism for prodrug activation, which could be leveraged to design novel prodrugs with high barriers to drug resistance.

microbiology↗