Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli

Abstract

Mecillinam (amdinocillin) is a β-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance. Results show that cysB mutations cause a gene regulatory response that changes the expression of ∼450 genes. Among the proteins that show increased levels are the PBP1B, LpoB, and FtsZ proteins, which are known to be involved in peptidoglycan biosynthesis. Artificial overexpression of either PBP1B or LpoB in a wild-type E. coli strain conferred mecillinam resistance; conversely, inactivation of either the mrcB gene (which encodes PBP1B) or the lpoB gene (which encodes the PBP1B activator LpoB) made cysB mutants susceptible. These results show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance. The addition of reducing agents to a cysB mutant converted it to full susceptibility, with associated downregulation of PBP1B, LpoB, and FtsZ. We propose a model in which cysB mutants confer mecillinam resistance by inducing a response that causes upregulation of the PBP1B and LpoB proteins. The higher levels of these two proteins can then rescue cells with mecillinam-inhibited PBP2. Our results also show how resistance can be modulated by external conditions such as reducing agents.

Cell density-dependent antibiotic tolerance to inhibition of the elongation machinery requires fully functional PBP1B

“…However, the ΔPBP1B cells stop growing within an hour and eventually lyse when spiked with A22 or mec (Figs. 5a–d and S 14 ), supporting previous reports 44 , 45 . These data suggest that PBP1B is needed for DDGR of both A22 and mec treatments.…”

“…5f ), is a way of overcoming DDGR. This is supported by both demonstrating peptidoglycan synthesis to have a division of labor between the divisome and elongasome and each having their own essential enzymes and proteins 5 , 16 , 37 , 45 A cocktail of mecillinam, a clinically approved antibiotic for use in humans that is not currently widely used, and cefsulodin or cephalexin could increase the efficacy of both while only using two antibiotics and minimizing drug interactions.…”

“…While PBP1A and PBP1B can compensate for each other, PBP1B does have distinct functionality from PBP1A. PBP1B’s transglycosylase domain is essential for de novo peptidoglycan synthesis, while PBP1A is unable to perform de novo peptidoglycan synthesis 44 , 45 , 48 . However, if either the transglycosylase domain or the transpeptidase domain are inhibited DDGR cannot occur.…”

Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1 , Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes

“…CysB is also known to regulate expression of diverse genes, such as those encoding acid tolerance ( 70 , 71 ), antibiotic resistance ( 72 , 73 ), type III secretion ( 74 ), and 4-hydroxy-2-alkylquinolones (HAQs). HAQs are core constituents of Pseudomonas aeruginosa cell-to-cell signaling that control various physiological functions, such as the secretion of extracellular DNA and biofilm formation ( 73 , 75 ).…”

Resistance to Mecillinam and Nine Other Antibiotics for Oral Use in Escherichia coli Isolated from Urine Specimens of Primary Care Patients in Germany, 2019/20

“…The cysB gene encodes for the CysB protein, which is the major positive regulator of the cysteine biosynthesis pathway. Thulin and Andersson showed that cysB mutations led to increased levels of the proteins PBP1B, LpoB, and FtsZ, which are known to be involved in peptidoglycan biosynthesis [ 27 ]. CysB-related mecillinam resistance, however, was only expressed in growth media with low concentrations of cysteine (including Mueller–Hinton broth) and cysB mutants returned to mecillinam susceptibility when the media were supplemented with cysteine.…”