Environmental community transcriptomics: strategies and struggles.

Transcriptomics is the study of RNA transcripts, the portion of the genome that is transcribed, in a specific cell, tissue, or organism. Transcriptomics provides insight into gene expression patterns, regulation, and the underlying mechanisms of cellular processes. Community transcriptomics takes this a step further by studying the RNA transcripts from environmental assemblies of organisms, with the intention of better understanding the interactions between members of the community. Community transcriptomics requires successful extraction of RNA from a diverse set of organisms and subsequent analysis via mapping those reads to a reference genome or de novo assembly of the reads. Both, extraction protocols and the analysis steps can pose hurdles for community transcriptomics. This review covers advances in transcriptomic techniques and assesses the viability of applying them to community transcriptomics.

Development of a Selective and Stable Antimicrobial Peptide.

Antimicrobial peptides (AMPs) are presented as potential scaffolds for antibiotic development due to their desirable qualities including broad-spectrum activity, rapid action, and general lack of susceptibility to current resistance mechanisms. However, they often lose antibacterial activity under physiological conditions and/or display mammalian cell toxicity, which limits their potential use. Identification of AMPs that overcome these barriers will help develop rules for how this antibacterial class can be developed to treat infection. Here we describe the development of our novel synthetic AMP, from discovery through in vivo application. Our evolved AMP, DTr18-dab, has broad-spectrum antibacterial activity and is nonhemolytic. It is active against planktonic bacteria and biofilm, is unaffected by colistin resistance, and importantly is active in both human serum and a Galleria mellonella infection model. Several modifications, including the incorporation of noncanonical amino acids, were used to arrive at this robust sequence. We observed that the impact on antibacterial activity with noncanonical amino acids was dependent on assay conditions and therefore not entirely predictable. Overall, our results demonstrate how a relatively weak lead can be developed into a robust AMP with qualities important for potential therapeutic translation.

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.

Designing and identifying ß-hairpin peptide macrocycles with antibiotic potential.

Peptide macrocycles are a rapidly emerging class of therapeutic, yet the design of their structure and activity remains challenging. This is especially true for those with ß-hairpin structure due to weak folding properties and a propensity for aggregation. Here, we use proteomic analysis and common antimicrobial features to design a large peptide library with macrocyclic ß-hairpin structure. Using an activity-driven high-throughput screen, we identify dozens of peptides killing bacteria through selective membrane disruption and analyze their biochemical features via machine learning. Active peptides contain a unique constrained structure and are highly enriched for cationic charge with arginine in their turn region. Our results provide a synthetic strategy for structured macrocyclic peptide design and discovery while also elucidating characteristics important for ß-hairpin antimicrobial peptide activity.

Identification of novel proteins in the Dictyostelium discoideum chemorepulsion pathway using REMI.

Chemorepulsion, the biased migration of a cell away from a signal, is essential for many biological processes and the ability to manipulate chemorepulsion could lead to new therapeutics for a variety of diseases. However, little is known about eukaryotic cell chemorepulsion. Utilizing the model organism Dictyostelium discoideum, we previously identified an endogenous chemorepellent protein secreted by D. discoideum cells called AprA, and proteins involved in the AprA-induced chemorepulsion pathway including the G protein-coupled receptor GrlH, G beta and G protein alpha 8 protein subunits, protein kinase A, components of the mammalian target of rapamycin complex 2 (mTORC2), phospholipase A, PTEN and a PTEN-like phosphatase (CnrN), a retinoblastoma orthologue (RblA), extracellular signal-regulated kinase 1 (Erk1), p-21 activated protein kinase D (PakD), and the Ras proteins RasC and RasG. In this report, we used a genetic screen to identify 17 additional proteins involved in the AprA-induced chemorepulsion pathway .

Complete Genome Sequence of Stenotrophomonas maltophilia Siphophage Suso.

Phage Suso is a temperate siphophage of Stenotrophomonas maltophilia with a 44,659-bp genome. This phage is closely related to Stenotrophomonas phage SM171, sharing 92% overall nucleotide identity as determined by BLASTn, and it shares 14 similar proteins (BLASTp, E value < 0.001) with some Pseudomonas phages from the genus Beetrevirus.

Complete Genome Sequence of Klebsiella aerogenes Myophage Metamorpho.

The bacterium Klebsiella aerogenes is an opportunistic pathogen that often infects hospitalized patients and those who are immunocompromised. K. aerogenes in some cases can become resistant to antibiotic treatment. Being a potential therapeutic, Metamorpho is a T4-like myophage targeting K. aerogenes.