Computationally Aided Discovery of LysEFm5 Variants with Improved Catalytic Activity and Stability.

Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant Enterococcus faecium (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (±0.4)-fold improvement in specific activity and an 11.5 ± 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties.IMPORTANCE Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophage-derived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants.

Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models.

Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C.IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

Multispecies activity screening of microcin J25 mutants yields antimicrobials with increased specificity toward pathogenic Salmonella species relative to human commensal Escherichia coli.

Modern large-scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic-resistant bacterial infections of humans with species of Salmonella being a leading agriculture-based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multiple Salmonella species. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off-target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25, Escherichia coli are the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity against Salmonella and E. coli strains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point-mutant study revealed that microcin J25I13T reduces off-target activity toward commensal human-derived E. coli isolates by 81% relative to Salmonella enterica serovar Enteritidis. These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts.

Prehospital pediatric King LT-D use: a pilot study.

OBJECTIVE: To determine whether prehospital providers can successfully place a pediatric King laryngeal tube (LT-D) and ventilate a Laerdal SimBaby pediatric simulator during a respiratory arrest simulation. METHODS: We studied the ability of 45 paramedics and flight nurses to place the pediatric King LT-D in a SimBaby manikin. For the purposes of this study, paramedics and flight nurses were considered equivalent, because in this air medical system they have the same scope of practice in regard to airway skills. Because the participants had previous training and field experience with the adult King LT-D, we limited pediatric King LT-D training to our standard adult training plus selecting the correct size and inflation volumes for the device. Outcomes included rate of successful pediatric King LT-D placement, number of attempts to correctly place the tube, and time to first adequate ventilation. The subjects were evaluated on airway management using an 11-point skill test. A score of 8 or greater (≥ 73%) was considered passing. The subjects indicated their perceptions and preferences for the pediatric King LT-D using a five-point Likert scale. Data were analyzed using descriptive statistics. RESULTS: Crew members successfully placed the pediatric King LT-D 95.5% (43/45) of the time. The median number of attempts was one. Four subjects required a second attempt; two of these subjects failed at placement. Mean time to placement was 34 seconds (95% confidence interval [CI]: 26.4-67.3 sec). Ninety percent of the participants (40/45) successfully completed the skill test, with a mean score of 78.2% (95% CI: 73.6-82.7). The subjects strongly agreed that their previous training on the adult King LT-D and using it in the field had adequately prepared them to use the pediatric King LT-D. The subjects agreed that the pediatric King LT-D was easier to place than a pediatric endotracheal tube; they strongly agreed that they would use the pediatric King LT-D as an alternative airway. The participants disagreed that they would prefer the pediatric King LT-D as a primary means of securing pediatric airways. CONCLUSIONS: The pediatric King LT-D was quickly and reliably placed. Providers perceived the pediatric King LT-D to be easier to use than pediatric endotracheal intubation in this setting.

Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.

Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast-protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.

A Platform for Deep Sequence-Activity Mapping and Engineering Antimicrobial Peptides.

Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum would be powerful to elucidate sequence-activity landscapes and identify potent mutants. Yet the complexity of antimicrobial activity has largely constrained the scope and mechanistic bandwidth of AMP variant analysis. We developed a platform to efficiently perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to intracellularly express AMPs, grown under selective pressure, and deep sequenced to quantify mutant depletion. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we mapped the sequence-activity landscape of 170 000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation supported the platform's sensitivity and accuracy. The mapped landscape revealed an extended oncocin pharmacophore contrary to earlier structural studies, clarified the C-terminus role in internalization, identified functional epistasis, and guided focused, successful synthetic peptide library design, yielding a mutant with 2-fold enhancement in both intracellular and extracellular activity. The efficiency of SAMP-Dep poises the platform to transform AMP engineering, characterization, and discovery.

Risk factors for unsuccessful prehospital laryngeal tube placement.

INTRODUCTION: Laryngeal tube (LT) airways are commonly used in the prehospital setting, but there are limited data on clinical success rates across emergency medical services (EMS) agencies. We aimed to determine factors associated with unsuccessful LT placement in the prehospital setting. METHODS: We retrospectively reviewed all King LT placement attempts by prehospital providers in 35 ground advanced life support EMS agencies and one air medical critical care service with 17 rotorwing bases, between January 1, 2006 and August 31, 2011. Success of King LT placement and patient, procedural, and agency factors present were identified using descriptive statistics. Factors associated with unsuccessful laryngeal tube placement were identified using multivariable logistic regression. RESULTS: During the study period, we observed 511 attempts at laryngeal tube placement by paramedics or prehospital nurses in 477 patients. Unsuccessful LT placement occurred in 15.1% of first attempts and 9.9% of cases overall. The majority (79.2%) of first attempts occurred as a rescue airway after unsuccessful endotracheal intubation attempt(s), in patients with non-traumatic complaints (70.9%) and in cardiac arrest (60.8%). Gag reflex (OR 4.08, 95% CI 1.72-9.67), ground (versus air) EMS agency (OR 2.49, 95% CI 1.07-5.79), and male gender (OR 1.90, 95% CI 1.04-3.46) were associated with unsuccessful LT placement in our multivariable model. CONCLUSION: The laryngeal tube is an effective airway management tool for both advanced life support and critical care prehospital providers. Gag reflex, ground (versus air) EMS agency, and male gender were associated with unsuccessful laryngeal tube placement by prehospital personnel.