Biology subjects

Ronning, D. R.

Publications and source records attributed to Ronning, D. R..

3 recordsLinked to original sources

bioRxiv2025

Structure-Guided Development of a Potent BioA Inhibitor ValidatesBiotin Synthesis Inhibition as a Therapeutic Strategy forTuberculosis

Conditionally essential pathways - such as the biotin biosynthesis - represent promising targets for new antibiotics. However, the chemical interrogation of the biotin pathway with an orally effective lead remains elusive, and the preclinical development of biotin inhibitors for mycobacterial infections in vivo is challenging due to the unusually high concentration of biotin in standard mouse models. Structure-guided optimization was applied to develop the first oral lead targeting aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis, resulting in C48, a picomolar inhibitor displaying sub-micromolar MICs against Mycobacterium tuberculosis (Mtb). Mechanism of action was confirmed by biochemical, structural, and genetic studies. C48 demonstrated favorable pharmacokinetics and excellent oral bioavailability resulting in over 39,000-fold improved exposure. We next developed an easy-to-operate, low-biotin mouse model that recapitulates human biotin physiology. C48 significantly reduced Mtb burden in this low-biotin mouse model, providing the first in vivo proof-of-concept for targeting biotin biosynthesis in Mtb.

microbiology↗

bioRxiv2025

A D-alanine aminotransferase S180F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation

Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of D-alanine for cell wall synthesis, the target of {beta}-lactam antibiotics. Furthermore cycA-dependent alanine transport controls MRSA {beta}-lactam susceptibility in chemically defined medium (CDM) in a glucose-dependent manner. Here we report that S. aureus was auxotrophic for L-alanine in CDM, and that this growth defect was rescued by glucose (or compensatory mutations), but only when the alanine racemase (alr1) and D-alanine aminotransferase (dat) genes were functional. No role was observed for the alanine dehydrogenase 1 (ald1) and ald2 genes. As previously reported, alr1 and, to a lesser extent, cycA mutations increased susceptibility to D-cycloserine (DCS). In contrast, only alr1 mutation increased susceptibility to {beta}-chloro-D-alanine (BCDA), suggesting distinct targets for these alanine analogue antibiotics, which act synergistically against MRSA. Genome sequencing of a BCDA-resistant mutant identified a C539T mutation in dat, predicted to result in a S180F substitution. Expression of the datC539T operon in wild-type increased BCDA resistance. alr1/dat::Em and alr1/datC539T double mutants were auxotrophic for D-alanine, indicating that Dat-S180F transaminase activity is impaired, a conclusion supported by in vitro enzyme assays. Structural modeling revealed an active-site loop shift in Dat-S180F that altered PLP co-factor binding. Molecular docking showed that the S180F substitution promotes BCDA-PLP adduct dissociation by releasing inactivated BCDA, thereby conferring resistance. These data reveal essential roles for Alr1 and Dat during growth under nutrient-limiting conditions and the potential of combination therapy separately targeting both enzymes with DCS and BCDA to extend the treatment options for MRSA infections.

microbiology↗

bioRxiv2024

Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus. We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus. Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a substantial intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication. SignificanceUnder mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus. We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus.

microbiology↗