Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories

Differential active site requirements for NDM-1 β-lactamase hydrolysis of carbapenem versus penicillin and cephalosporin antibiotics.

New Delhi metallo-β-lactamase-1 exhibits a broad substrate profile for hydrolysis of the penicillin, cephalosporin and 'last resort' carbapenems, and thus confers bacterial resistance to nearly all β-lactam antibiotics. Here we address whether the high catalytic efficiency for hydrolysis of these diverse substrates is reflected by similar sequence and structural requirements for catalysis, i.e., whether the same catalytic machinery is used to achieve hydrolysis of each class. Deep sequencing of randomized single codon mutation libraries that were selected for resistance to representative antibiotics reveal stringent sequence requirements for carbapenem versus penicillin or cephalosporin hydrolysis. Further, the residue positions required for hydrolysis of penicillins and cephalosporins are a subset of those required for carbapenem hydrolysis. Thus, while a common core of residues is used for catalysis of all substrates, carbapenem hydrolysis requires an additional set of residues to achieve catalytic efficiency comparable to that for penicillins and cephalosporins

Novel Mechanism for Nisin Resistance via Proteolytic Degradation of Nisin by the Nisin Resistance Protein NSR▿

Nisin is a 34-residue antibacterial peptide produced by Lactococcus lactis that is active against a wide range of gram-positive bacteria. In non-nisin-producing L. lactis, nisin resistance could be conferred by a specific nisin resistance gene (nsr), which encodes a 35-kDa nisin resistance protein (NSR). However, the mechanism underlying NSR-mediated nisin resistance is poorly understood. Here we demonstrated that the protein without the predicted N-terminal signal peptide sequence, i.e., NSRSD, could proteolytically inactivate nisin in vitro by removing six amino acids from the carboxyl “tail” of nisin. The truncated nisin (nisin1-28) displayed a markedly reduced affinity for the cell membrane and showed significantly diminished pore-forming potency in the membrane. A 100-fold reduction of bactericidal activity was detected for nisin1-28 in comparison to that for the intact nisin. In vivo analysis indicated that NSR localized on the cell membrane and endowed host strains with nisin resistance by degrading nisin as NSRSD did in vitro, whereas NSRSD failed to confer resistance upon the host strain. In conclusion, we showed that NSR is a nisin-degrading protease. This NSR-mediated proteolytic cleavage represents a novel mechanism for nisin resistance in non-nisin-producing L. lactis

Bovicin HJ50-Like Lantibiotics, a Novel Subgroup of Lantibiotics Featured by an Indispensable Disulfide Bridge

<div><p>Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by <i>Streptococcus bovis</i> HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel <i>lan</i> loci from <i>Clostridium perfringens</i> D str. JGS1721, <i>Bacillus cereus</i> As 1.348 and <i>B. thuringiensis</i> As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.</p></div

Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation

Mutation, substitution and alkylation of disulfide bridge in bovicin HJ50-like lantibiotics.

<p>(A) Antimicrobial activity of wild type bovicin HJ50 (25 µL, 5 µg/mL) when treated respectively with 0, 2, 4, 8 mM TCEP. (B) MS analysis of purified NEM-alkylated bovicin HJ50. (C) Antimicrobial activity of wild type bovicin HJ50 (WT) and alkylated bovicin HJ50 (WT-NEM). (D) Antimicrobial activity of disulfide-related mutants. D/K represents substitution of disulfide-forming Cys residues to Asp and Lys, e.g., bovicin HJ50 D/K means bovicin HJ50 mutant C21D/C29K. L/L or F/F is referred as the same condition as D/K. 25 µL peptide samples of 20 µg/mL were applied to each hole.</p