Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase-mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, while Lys 234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a five-fold increase in kcat for ampicillin, suggesting the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.

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Norovirus (NoV) infections are a leading cause of gastroenteritis. The humoral immune response plays an important role in the control of NoV and recent studies have identified neutralizing antibodies that bind the capsid protein VP1 to block viral infection. Here, we utilize a NoV GI.1 Jun-Fos assisted phage display library constructed from randomly fragmented genomic DNA coupled with affinity selection for antibody binding and subsequent deep sequencing to map epitopes. The epitopes were identified by quantitating the enrichment of phage clones before and after affinity selection and aligning the sequences of the most enriched peptides. The HJT-R3-A9 scFv antibody epitope was mapped to a 12 amino acid region of VP1 that is also the binding site of several previously identified mAbs. We synthesized the 12-mer peptide and found it binds the scFv antibody with a KD of 46 nM. Further, alignment of enriched peptides after affinity selection on rabbit anti-NoV polyclonal sera revealed five families of overlapping sequences that define distinct epitopes in VP1. One of these is identical to the HJT-R3-A9 scFv epitope, further suggesting it is immunodominant. Similarly, other epitopes identified using the polyclonal sera overlap binding sites from previously reported mAbs, suggesting they are also dominant epitopes. The results demonstrate that affinity selection and deep sequencing of the phage library provides sufficient resolution to map multiple epitopes simultaneously from complex samples such as polyclonal sera. This approach can be extended to examine the antigenic landscape in patient sera to facilitate investigation of the immune response to NoV. IMPORTANCE Norovirus (NoV) infections are a leading cause of gastroenteritis in the United States. Human noroviruses exhibit extensive genetic and antigenic diversity, which makes it challenging to design a vaccine that provides broad protection against infection. Antibodies developed during the immune response play an important role in the control of NoV infections. Neutralizing antibodies have been identified that act by sterically blocking the site on the virus used to bind human cells. Identification of other antibody binding sites associated with virus neutralization is therefore of interest. Here we use a high resolution method to map multiple antibody binding sites simultaneously from complex sera samples. The results show a relatively small number of sites on the virus bind a large number of independently generated antibodies, suggesting immundominance plays a role in the humoral immune response to NoV infections.

Protein−protein interactions govern many cellular processes, and identifying binding interaction sites on proteins can facilitate the discovery of inhibitors to block such interactions.Here we identify peptides from a randomly fragmented plasmid encoding the β-lactamase inhibitory protein (BLIP) and the Lac repressor (LacI) that represent regions of protein−protein interactions. We utilized a Jun-Fos-assisted phage display system that has previously been used to screen cDNA and genomic libraries to identify antibody antigens. Affinity selection with polyclonal antibodies against LacI or BLIP resulted in the rapid enrichment of in-frame peptides from various regions of the proteins. Further, affinity selection with β-lactamase enriched peptides that encompass regions of BLIP previously shown to contribute strongly to the binding energy of the BLIP/β-lactamase interaction, i.e., hotspot residues. Further, one of the regions enriched by affinity selection encompassed a disulfide-constrained region of BLIP that forms part of the BLIP interaction surface in the native complex that we show also binds to β-lactamase as a disulfideconstrained macrocycle peptide with a K D of ∼1 μM. Fragmented open reading frame (ORF) libraries may efficiently identify such naturally constrained peptides at protein−protein interaction interfaces. With sufficiently deep coverage of ORFs by peptide-coding inserts, phage display and deep sequencing can provide detailed information on the domains or peptides that contribute to an interaction. Such information should enable the design of potentially therapeutic macrocycles or peptidomimetics that block the interaction.

Carbapenemases confer resistance to nearly all β-lactam antibiotics. The extensive spread of carbapenemase-producing multidrug-resistant bacteria contributes significantly to hospital-acquired infections. We have developed a novel protein-based binding assay that identifies KPC β-lactamases from clinical isolates. We used the protein-protein interaction between KPCs and a soluble β-lactamase inhibitory protein (BLIP) variant, BLIPK74T/W112D, which specifically inhibits KPCs but not other β-lactamases. In this assay, BLIPK74T/W112D was allowed to form complexes with KPC-2 in bacterial cell lysates and then extracted using His tag binding resins. We demonstrated the presence of KPC-2 by monitoring the hydrolysis of a colorimetric β-lactam substrate. Also, to further increase the accuracy of the method, a BLIPK74T/W112D-mediated inhibition assay was developed. The binding and inhibition assays were validated by testing 127 Klebsiella pneumoniae clinical isolates with known genome sequences for the presence of KPC. Our assays identified a total of 32 strains as KPC-2 producers, a result in 100% concordance with genome sequencing predictions. To further simplify the assay and decrease the time to obtain results, the BLIPK74T/W112D protein was tested in combination with the widely used Carba-NP assay. For this purpose, the genome-sequenced K. pneumoniae strains were tested for the presence of carbapenemases with the Carba-NP test with and without the addition of BLIPK74T/W122D. The test accurately identified carbapenemase-producing strains and the addition of BLIPK74T/W112D allowed a further determination that the strains contain KPC carbapenemase. Thus, the BLIPK74T/W112D protein is an effective sensor to specifically detect KPC β-lactamases produced by clinical isolates. IMPORTANCE Infections caused by carbapenem-resistant Enterobacteriaceae are associated with high therapeutic failure and mortality rates. Thus, it is critical to rapidly identify clinical isolates expressing KPC β-lactamases to facilitate administration of the correct antibiotic treatment and initiate infection control strategies. To address this problem, we developed a protein-based, KPC-specific binding assay in combination with a cell lysate inhibition assay that provided a 100% identification rate of KPC from clinical isolates of known genomic sequence. In addition, this protein sensor was adapted to the Carba-NP assay to provide a rapid strategy to detect KPC-producing isolates that will facilitate informed treatment of critically ill patients.