Adapting antibacterial display to identify serum active macrocyclic peptide antibiotics.

The lack of available treatments for many antimicrobial resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity towards human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of gram-negative bacteria without strong inner membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo , suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality. Significance: Traditional methods of natural antibiotic discovery are low throughput and cannot keep pace with the development of antimicrobial resistance. Synthetic peptide display technologies offer a high-throughput means of screening drug candidates, but rarely consider functionality beyond simple target binding and do not consider retention of function in vivo . Here, we adapt a function-based, antibacterial display technology to screen a large library of peptide macrocycles directly for bacterial growth inhibition in human serum. This screen identifies an optimized non-toxic macrocyclic peptide antibiotic retaining in vivo function, suggesting this advancement could increase clinical antibiotic discovery efficiency.

Adapting antibacterial display to identify serum-active macrocyclic peptide antibiotics.

The lack of available treatments for many antimicrobial-resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity toward human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of Gram-negative bacteria without strong inner-membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

Generation of Synthetic Acinetobacter baumannii-Specific Nanobodies.

The bacterial pathogen Acinetobacter baumannii is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that can recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next-generation sequencing analysis can aid in the selection of lead candidate nanobodies. Using monoclonal phage display, we validated the binding of lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that specifically bound select A. baumannii strains compared to other common drug-resistant pathogens. These findings support the potential for nanobodies to selectively target A. baumannii and the identification of lead candidates for future investigation.

Using display technologies to identify macrocyclic peptide antibiotics.

Antibiotic resistant bacterial infections are now a leading cause of global mortality. While drug resistance continues to spread, the clinical antibiotic pipeline has become bare. This discord has focused attention on developing new strategies for antimicrobial discovery. Natural macrocyclic peptide-based products have provided novel antibiotics and antibiotic scaffolds targeting several essential bacterial cell envelope processes, but discovery of such natural products remains a slow and inefficient process. Synthetic strategies employing peptide display technologies can quickly screen large libraries of macrocyclic sequences for specific target binding and general antibacterial potential providing alternative approaches for new antibiotic discovery. Here we review cell envelope processes that can be targeted with macrocyclic peptide therapeutics, outline important macrocyclic peptide display technologies, and discuss future strategies for both library design and screening.

Discriminating arboviral species.

Mosquito-borne arboviruses, including a diverse array of alphaviruses and flaviviruses, lead to hundreds of millions of human infections each year. Current methods for species-level classification of arboviruses adhere to guidelines prescribed by the International Committee on Taxonomy of Viruses (ICTV), and generally apply a polyphasic approach that might include information about viral vectors, hosts, geographical distribution, antigenicity, levels of DNA similarity, disease association and/or ecological characteristics. However, there is substantial variation in the criteria used to define viral species, which can lead to the establishment of artificial boundaries between species and inconsistencies when inferring their relatedness, variation and evolutionary history. In this study, we apply a single, uniform principle - that underlying the Biological Species Concept (BSC) - to define biological species of arboviruses based on recombination between genomes. Given that few recombination events have been documented in arboviruses, we investigate the incidence of recombination within and among major arboviral groups using an approach based on the ratio of homoplastic sites (recombinant alleles) to non-homoplastic sites (vertically transmitted alleles). This approach supports many ICTV-designations but also recognizes several cases in which a named species comprises multiple biological species. These findings demonstrate that this metric may be applied to all lifeforms, including viruses, and lead to more consistent and accurate delineation of viral species.

Recombination events are concentrated in the spike protein region of Betacoronaviruses.

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.