Mechanism and Diversity of the Erythromycin Esterase Family of Enzymes

Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of <i>Bacillus cereus</i>, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections

Antibiotic Resistance Is Prevalent in an Isolated Cave Microbiome

<div><p>Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.</p> </div

Summary of Antibiotic Inactivation Studies for Gram-Negative Isolates.

<p>Strains were grown in 50% TSB for 5 days in presence of 20 µg/ml antibiotic. Conditional media was used for setting up disk diffusion assays and LC-MS analyses. Inactivation was defined as the absence of a zone of clearance around the disk. Hydrolytic mechanism of ß-lactam resistance is inferred.</p

Kinetic parameters for MPH (2′)-II from <i>B. paraconglomeratum.</i>

<p>1- GTP held at 200 µM for antibiotic substrates, 2-erythomycin held at 400 µM.</p

Induction of daptomycin resistance in <i>Paenibacillus lautus</i> LC231.

<p>LC231 was cultured in TSB supplemented with 1.25 mM CaCl₂ and 4 µg/ml daptomycin added from start (zero time point) (1) or early log phase (2). Growth was compared to growth control with no drug (3). Arrow represents the time point at which daptomycin was added during early log phase.</p

MIC of antibiotics determined in this study.

<p>Heat Plot for (A) Gram-positive strains (B) Gram-negative strains against various antibiotics. Antibiotics are grouped according to their mode of action and the gradient from light blue to dark blue represents the range from lowest MIC value (0.3 µg/ml) to highest MIC value (256 µg/ml) as shown in the legend. White means no MIC was determined.</p

Summary of Antibiotic Inactivation Studies for Gram-positive Isolates.

<p>Strains were grown in 50% TSB for 5 days in presence of 20 µg/ml antibiotic. Conditional media was used for setting up disk diffusion assays and LC-MS analyses. Inactivation was defined as the absence of a zone of clearance around the disk. Hydrolytic mechanism of ß-lactam resistance is inferred.</p

Resistance levels of Lechuguilla cave bacteria at 20 µg/ml against various antibiotics: (top) Gram-positive strains (bottom) Gram-negative strains.

<p>Antibiotics are grouped according to their mode of action/target, where each color represents a different target.</p

Kinetic parameters for MPH (2′)-II from <i>B. faecium</i> DSM 4810.

<p>1- GTP held at 200 µM for antibiotic substrates, 2-erythomycin held at 400 µM.</p

Plan and profile maps of Lechuguilla Cave, Carlsbad Caverns National Park, New Mexico.

<p>The sites where microbial strains were collected (LCECE, LCDS1 and LCEA1) are shown relative to the entrance and depth. tN represents true North on the plan, while the profile has an exaggerated vertical profile of 1.5×.</p