Recently, two related Streptococcus pyogenes strains with reduced susceptibility to ampicillin, amoxicillin, and cefotaxime, antibiotics commonly used to treat S. pyogenes infections, were reported. The two strains had the same nonsynonymous (amino acid-substituting) mutation in the pbp2x gene, encoding penicillin-binding protein 2X (PBP2X). This concerning report led us to investigate our library of 7,025 genome sequences of type emm1, emm28, and emm89 S. pyogenes clinical strains recovered from intercontinental sources for mutations in pbp2x. We identified 137 strains that, combined, had 37 nonsynonymous mutations in 36 codons in pbp2x. Although to a lesser magnitude than the two previously published isolates, many of our strains had decreased susceptibility in vitro to multiple beta-lactam antibiotics. Many pbp2x mutations were found only in single strains, but 16 groups of two or more isolates of the same emm type had an identical amino acid replacement. Phylogenetic analysis showed that, with one exception, strains of the same emm type with the same amino acid replacement were clonally related by descent. This finding indicates that strains with some amino acid changes in PBP2X can successfully spread to new human hosts and cause invasive infections. Mapping of the amino acid changes onto a three-dimensional structure of the related Streptococcus pneumoniae PBP2X suggests that some substitutions are located in regions functionally important in related pathogenic bacterial species. Decreased beta-lactam susceptibility is geographically widespread in strains of numerically common emm gene subtypes. Enhanced surveillance and further epidemiological and molecular genetic study of this potential emergent antimicrobial problem are warranted.
Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.
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.
Disrupting protein‐protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP‐II proteins are unrelated in sequence and structure and yet each potently inhibit β‐lactamases. High‐throughput oligonucleotide synthesis was used to construct a 12,470‐member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP‐II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with β‐lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP‐II contact residues in the binding interface occurred. BLIP and BLIP‐II peptides that were enriched by affinity selection were shown to bind β‐lactamases and disrupt the BLIP/β‐lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.