b Mutation-dependent overproduction of intrinsic ␤-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the ␤-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on ␤-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gramnegative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious ␤-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic ␤-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are >1.5% in the clinical setting. Pseudomonas aeruginosa is a well-known cause of acute and chronic infections in fragile patients. One of the most remarkable traits of this opportunistic pathogen is its ability to evolve and become resistant to many antibiotics through a variety of mutational and transferable mechanisms (reviewed in reference 1). Some mechanisms tend to prevent the interaction of drugs with their cognate cellular targets, while others result in drug inactivation (1). Like several other Gram-negative species, P. aeruginosa harbors a chromosomal drug-inducible gene, bla AmpC , encoding a wide-spectrum class C ␤-lactamase (2). This enzyme contributes to the natural resistance of the microorganism toward labile and inducing molecules, such as aminopenicillins, first-and secondgeneration cephalosporins (3). More importantly, when overproduced as a result of mutations altering the peptidoglycan recycling process, AmpC becomes a major cause of resistance to widely used antipseudomonal penicillins (ticarcillin and piperacillin), monobactams (aztreonam), and third-generation (ceftazidime) and fourth-generation (cefepime) cephalosporins (4-7). The so-called "derepressed mutants" are common in the clinical setting and account for a large proportion of strains resistant to ceftazidime and cefepime in various studies (8-11)....

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.