kinact / KI Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are inhibited by the ß-lactam class of antibiotics. PBP inhibition disrupts cell wall biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC 50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity but can be misleading in the study of time-dependent, irreversible inhibitors. Due to this disconnect, the second order rate constant k inact / K I is a more appropriate metric of covalent inhibitor potency. Despite being the gold standard measurement of potency, k inact / K I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologs (such as the PBPs). Therefore, we developed a whole-cell k inact / K I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent activity-based probe Bocillin-FL. Our results align with in vitro k inact / K I data and show a comparable relationship to previously established IC 50 values. These results support the validity of our in vivo k inact / K I method as a means of obtaining a full picture of ß-lactam potency for a suite of PBPs.

Failure of mecA/mecC PCR Testing to Accurately Predict Oxacillin Resistance in a Patient with Staphylococcus aureus Infective Endocarditis.

Genotypic testing for mecA/mecC is heavily relied upon for rapid optimization of antimicrobial therapy in infections due to Staphylococcus aureus. Little is known regarding optimal reporting and/or therapy for patients demonstrating lack of genotypic evidence of mecA or mecC but phenotypic oxacillin resistance. We report a case of a 77-year-old patient with S. aureus bloodstream infection and infective endocarditis with discordance between mecA/mecC genotypic results and phenotypic susceptibility testing.

Current regulatory landscape for viral point-of-care testing in the United States.

Historically, the diagnosis of viral infections has been accomplished using a combination of laboratory-based methods, including culture, serology, antigen-based tests, and molecular (e.g., real-time PCR) assays. Although these methods provide an accurate way to detect viral pathogens, testing in a centralized laboratory may delay results, which could impact patient diagnosis and management. Point-of-care tests, including antigen- and molecular-based assays, have been developed to assist with the timely diagnosis of several viral infections, such as influenza, respiratory syncytial virus, and COVID-19. Despite the ability of point-of-care tests to provide rapid results (i.e., <30 min), there are issues to consider prior to their routine use, including test performance and specific regulatory requirements. This review will provide a summary of the regulatory landscape of point-of-care tests for viral infections in the United States, and address important considerations such as site certification, training and inspection readiness.

Leptotrichia Bacteremia: 10-Year Retrospective Clinical Analysis and Antimicrobial Susceptibility Profiles.

Leptotrichia species are anaerobic, Gram-negative bacilli increasingly recognized as pathogens capable of causing invasive infections such as bloodstream infection (BSI), particularly among immunocompromised patients. However, there is a paucity of data regarding epidemiology, antimicrobial susceptibility, optimal treatment, and clinical outcomes among patients with Leptotrichia bacteremia. Patient risk factors, treatment approaches, and outcomes of a retrospective cohort of adult patients with Leptotrichia BSI at a tertiary medical center (Mayo Clinic Rochester [MCR]) were evaluated. Concurrently, species, temporal trends, and antimicrobial susceptibility testing (AST) results of Leptotrichia isolates submitted to a reference laboratory (Mayo Clinic Laboratories) over the past 10 years were examined. We identified 224 blood culture isolates of Leptotrichia species, with 26 isolates from patients treated at MCR. The most frequent species included L. trevisanii (49%), L. buccalis (24%), and L. wadei (16%). Leptotrichia species demonstrated >90% susceptibility to penicillin, metronidazole, ertapenem, and piperacillin-tazobactam. However, 96% (74/77) of isolates were resistant to moxifloxacin. For patients treated at MCR, the mean patient age was 55 years (standard deviation [SD], 17), with 9 females (35%), and all were neutropenic at the time of BSI. The primary sources of infection were gastrointestinal (58%), intravascular catheter (35%), and odontogenic (15%). Patients were treated with metronidazole (42%), piperacillin-tazobactam (27%), or carbapenems (19%). The mean duration of treatment was 11 days (SD, 4.5), with a 60-day all-cause mortality of 19% and no microbiologic relapse. Leptotrichia species are rare but important causes of BSI in neutropenic patients. Due to evolving antimicrobial susceptibility profiles, a review of AST results is necessary when selecting optimal antimicrobial therapy.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with ß-Lactones.

ß-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to ß-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of ß-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four ß-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the ß-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655.

Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of ß-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten ß-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal ß-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Enzyme-targeted fluorescent small-molecule probes for bacterial imaging.

Molecular imaging methods to visualize myriad biochemical processes in bacteria have traditionally been dependent upon molecular biology techniques to incorporate fluorescent biomolecules (e.g., fusion proteins). Such methods have been instrumental in our understanding of how bacteria function but are not without drawbacks, including potential perturbation to native protein expression and function. To overcome these limitations, the use of fluorescent small-molecule probes has gained much attention. Here, we highlight examples from the recent literature that showcase the utility of small-molecule probes for the fluorescence imaging of bacterial cells, including electrophilic, metabolic, and enzyme-activated probes. Although the use of these types of compounds for bacterial imaging is still relatively new, the selected examples demonstrate the exciting potential of these critical tools in the exploration of bacterial physiology.

Chemical tools for selective activity profiling of bacterial penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the ß-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4-18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a ß-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent ß-lactone probes.

Activity-Based Protein Profiling Methods to Study Bacteria: The Power of Small-Molecule Electrophiles.

ABPP methods have been utilized for the last two decades as a means to investigate complex proteomes in all three domains of life. Extensive use in eukaryotes has provided a more fundamental understanding of the biological processes involved in numerous diseases and has driven drug discovery and treatment campaigns. However, the use of ABPP in prokaryotes has been less common, although it has gained more attention over the last decade. The urgent need for understanding bacteriophysiology and bacterial pathogenicity at a foundational level has never been more apparent, as the rise in antibiotic resistance has resulted in the inadequate and ineffective treatment of infections. This is not only a result of resistance to clinically used antibiotics, but also a lack of new drugs and equally as important, new drug targets. ABPP provides a means for which new, clinically relevant drug targets may be identified through gaining insight into biological processes. In this chapter, we place particular focus on the discussion of ABPP strategies that have been applied to study different classes of bacterial enzymes.

Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae.

Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The ß-lactam antibiotics, which are among the most clinically effective antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified ß-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.