A neglected and emerging antimicrobial resistance gene encodes for a serine-dependent macrolide esterase.

The discovery of unreported antimicrobial resistance genes (ARGs) remains essential. Here, we report the identification and preliminary characterization of an α/ß-hydrolase that inactivates macrolides. This serine-dependent macrolide esterase co-occurs with emerging ARGs in the environment, animal microbiomes, and pathogens.

Structural and functional analysis of a bile salt hydrolase from the bison microbiome.

The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them as targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSHAc). Steady-state kinetic analyses demonstrated that BSHAc is a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second-order rate constants (kcat/KM) for BSHAc-catalyzed reactions of relevant bile salts-glyco- and tauro-conjugates of cholic acid and DCA- varied by ∼30-fold and measured between 1.4 × 105 and 4.3 × 106 M-1s-1. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHAc with a rate of inactivation (kinact) of ∼2 h-1 and a second order rate constant (kinact/KI) of ∼24 M-1s-1 for the process. Structural characterization of BSHAc reacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs.

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

Macrolide esterases: current threats and opportunities.

Antibiotics often contain ester bonds. The macrocyclic lactones of macrolides are pre-eminent examples in which ester bonds are essential to the form and function of antibiotics. Bacterial macrolide esterases that hydrolyze these macrocyclic lactones to confer antimicrobial resistance (AMR) are the topic of this forum. We provide insight into their role in agricultural systems and discuss their emergence and their potential extensibility to bioremediation efforts.