First Penicillin-Binding Protein Occupancy Patterns for 15 β-Lactams and β-Lactamase Inhibitors in Mycobacterium abscessus

Abstract

Mycobacterium abscessus (Mab) causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of Mab infections and the multitude of combinations that have been used clinically have had low success rates and high rates for toxicities. With β-lactam antibiotics being safe, double β-lactam and β-lactam/β-lactamase inhibitor combinations are of interest for improving treatment of Mab infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different β-lactams in Mab. We determined the preferred PBP targets of 13 β-lactams and two β-lactamase inhibitors in two Mab strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the β-lactam concentrations (IC50) that half-maximally inhibited Bocillin binding. Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016-0.5mg/L; cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031-1mg/L; ceftriaxone and cefotaxime) or intermediate concentrations (0.35-16mg/L; ceftazidime and cefoxitin). Sulbactam, aztreonam, carumonam, mecillinam and avibactam (cluster 3) inactivated the same PBPs as cephalosporins, but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2-16mg/L. Carbapenems, ceftriaxone and cefotaxime were the most promising β-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in Mab provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with β-lactams.

Combating Multidrug‐Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling

“…Proteomics provides information directly at the protein level using samples of complex mixtures and was critical to identify PBPs in K. pneumoniae 90 and Mycobacterium abscessus. 95,96 Thus, proteomics approaches are critical when determining PBP binding in previously unstudied pathogens.…”

“…90,99 Later, we found a modern version of a French pressure cell (model FBG12800, Pressure Cell Homogenizer (up to 60,000 psi); Stansted Fluid Power, Harlow, Essex, UK) to be more efficient and provide considerably better signal to noise ratios. 96 With either of these systems, the minimum volume of the bacterial suspension to be lysed is ~ 10-15 mL. A laser scanner (Amersham Typhoon RGB) 90,96 provided substantially better sensitivity and signal to noise ratios than a chemiluminescence scanner; the latter could not detect lowly expressed PBPs.…”

“…96 With either of these systems, the minimum volume of the bacterial suspension to be lysed is ~ 10-15 mL. A laser scanner (Amersham Typhoon RGB) 90,96 provided substantially better sensitivity and signal to noise ratios than a chemiluminescence scanner; the latter could not detect lowly expressed PBPs.…”

“…Any βlactam that requires high (i.e., supra-physiological) concentrations in a lysed cell binding assay will not be a clinically useful binder for the respective PBP. 96 These PBP binding studies may be affected by the presence of excessive βlactamase activity in membrane fractions, which can cause hydrolysis of Bocillin TM FL. This can be minimized by additional washing steps or by selective inactivation of the respective βlactamase gene(s).…”

“…This allows proteomics to definitively identify and quantify proteins and their post‐translational modifications based on unique peptide sequences that contain as few as 5 to 20 amino acids from digested proteins. Proteomics provides information directly at the protein level using samples of complex mixtures and was critical to identify PBPs in K. pneumoniae 90 and Mycobacterium abscessus 95,96 . Thus, proteomics approaches are critical when determining PBP binding in previously unstudied pathogens.…”