Curve fitting was performed using spline interpolation. For all experiments, lines and shaded regions indicate means and SD, respectively. A radiolabeled phosphate release assay Fig. Treatment with colistin causes slower phosphate leakage. Drug-resistant A. We performed the same experiment for a sensitive strain of A. However, A. Clear membrane disruption was observed in E.
Similar observations were recorded for Shigella flexneri MTCC , which showed membrane disruption followed by the loss of cytoplasmic contents fig. These results indicate that large-scale membrane damage is not a prerequisite for bacterial death, which occurs on much shorter time scales.
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In addition, pharmacokinetic experiments were performed Fig. At different time points, blood from individual mice was extracted via terminal cardiac puncture. From previous time-kill experiments performed in ex vivo whole human blood Fig. Differentially expressed genes DEGs displaying a 1.
A graphical representation of the features and relationships between GO terms is provided in fig. The molecular response is depicted in Fig. Genes associated with 67 inner membrane proteins and 4 outer membrane proteins were found to be significantly up-regulated, and only 4 membrane-associated genes were down-regulated. Up- and down-regulated GO-up and Go-down genes are colored green and red, respectively. For clarity, only DEGs belonging to GO terms with biological functions relevant to this study are shown. Note that some genes can have multiple functions and belong to multiple GO terms.
Note that some genes do not have corresponding gene names assigned. In these cases, the truncated Agilent ID has been used. Twenty-two membrane-associated genes belonging to membrane transport proteins were up-regulated. Fourteen membrane-associated genes belonging to electron transport chain components—NADH reduced form of nicotinamide adenine dinucleotide dehydrogenase seven genes , adenosine triphosphate ATP synthase three genes , and anaerobic electron transport components four genes —were up-regulated.
Other genes belonging to diverse metabolic pathways were also found to be up-regulated, hinting at metabolic inhibitory processes. Four genes ompR , ttg2C , nlpD , and lolC responsible for maintaining outer membrane integrity and three genes mraY , ispU , and pal responsible for cell wall synthesis were also up-regulated. Twelve genes belonging to ribosomal proteins 5 genes for 50 S subunit and 7 genes for 30 S subunit were up-regulated.
Six genes involved with translation, and four genes associated with other ribosome-associated processes, were also up-regulated. Further, eight genes associated with the citric acid cycle were also up-regulated. Alternatively, AMPs are known to trap ribosomal release factors 29 , inhibit ribosomal protein synthesis 27 , 28 , and cause ribosomal aggregation In these cases, the contributions of these genes to the molecular response of A.
Additional correlations may have been obscured because of spectral artifacts close to the diagonal. However, the lack of adequate nonsequential NOEs and poor chemical shift dispersion prevented the calculation of the solution structure in this condition. The labels indicate the residue numbers of the H N resonances involved in the correlation. E Summary of the NOE and chemical shift data used for assigning the secondary structure as a function of residue number.
F Ensemble of 30 calculated structures left and view down the helix axis right. The side-chain colors indicate hydrophobic white , acidic red , and basic blue residues. The spectral characteristics in the presence of DPC showed a marked improvement over the sample in methanol, with improved chemical shift dispersion, as well as improved transfer of the NOE. The first three rows depict sequential NOE connectivities, where the thickness of the bar indicates a weak, medium, or strong NOE.
Antimicrobial peptides under clinical investigation
The next row depicts the CSI. An ensemble of 30 structures was calculated using this NOE and chemical shift data [ Fig. All backbone dihedral angles fell within the favorable region of the Ramachandran plot fig. Figure 7F also depicts a view down the helix axis, with hydrophobic residues colored white Ala, Gly, Ile, Leu, and Phe , negatively charged residues colored red Glu , and positively charged residues colored blue Lys. It is evident that the residues are not evenly distributed but are clustered on the basis of their hydrophobicity.
Restraints used for structure calculation and structure statistics are summarized in table S9. The electrostatic surface potential and 3D hydrophobic moment 3D-HM vector calculated for the first model of the ensemble are shown in fig. The angle between the helix axis and the hydrophobic moment vector is The proliferation of drug-resistant pathogens over the past few decades has created an urgent need for new antibiotics.
The ESKAPE pathogen family is particularly concerning, as its members are increasingly displaying resistance to carbapenem class antibiotics. Treatment options for carbapenem-resistant infections are limited to drugs of last resort such as tigecycline and colistin. Unfortunately, both drugs have significant drawbacks: The prevalence of tigecycline resistance continues to increase 14 , while colistin displays nephrotoxic effects after prolonged treatment 16 — In this study, we used a maximum common subgraph computational approach to design AMPs Fig.
Residues were reduced to nodes, while intra-residue covalent and backbone hydrogen bonds were reduced to connecting edges. We then attempted to design new AMPs by maximizing the number of subgraphs shared between a given design and existing AMPs.
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Our method was based on the hypothesis that commonly occurring structural subgraphs would be evolutionarily conserved only if they bestowed their parent peptides with greater antimicrobial activity. Both are short lysine-rich peptides with a similar charge and hydrophobicity profile. The extreme differences in their cumulative toxicities must therefore arise from subgraphical or structural differences. Other AMPs also have toxicological characteristics that render them unsuitable for internal administration.
For example, gramicidin S 46 , melittin 47 , and lactoferrampin 48 display hemolytic properties. These results are interesting as colistin is known to be nephrotoxic across diverse patient cohorts. Recent studies have shown that colistin usage caused acute kidney injury in Besides toxicity, rapid elimination kinetics also limit the clinical use of AMPs.
A clinical dose of colistin remains above MIC concentrations in mouse serum for min 57 but only produces a fold reduction in CFU over 60 min Fig. Similarly, a clinical dose of meropenem remains above MIC concentrations in the serum of human volunteers for 5 hours 58 but only displays a fold CFU reduction in 4 hours To ensure the reproducibility of our results, A.
We have also deposited A. Genes for small-molecule transporters were also found to be significantly up-regulated upon treating A.
Membrane-interacting peptides have previously been shown to be amphipathic in nature 60 , having one hydrophobic and one hydrophilic surface. The 3D-HM vector indicates the most likely membrane-interacting surface of the peptide fig. Assuming a horizontally oriented planar membrane, the orientation shown in fig. S12B therefore represents the likely orientation of the peptide with respect to the membrane as predicted by the 3D-HM vector.
Because the angle between the calculated vector and the helix axis is only All peptides synthesized for this study were purchased from GenScript Inc. MICs were determined by the microwell dilution method, as described by Wiegand et al.
“Stapled” antimicrobial peptides could combat antibiotic resistance
This method was used to determine the efficacy of our peptides against a panel of 30 type cultures table S2. Most of our clinical isolates displayed mucoid or plaque morphologies, and growth could not be assayed using optical density readings. Instead, a modified protocol using resazurin was used to determine the efficacy of our peptides against 64 clinical isolates Table 2.
Resazurin, a weakly fluorescent dye, is reduced to fluorescent resorufin in direct proportion to microbial aerobic respiration. Thirty microliters of a 0. Because aerobic respiration also occurs in bacteriostatic cultures, the lack of aerobic respiration indicates bactericidal activity. Therefore, the resazurin assay measures the MBC. Experiments were performed to determine whether the designed peptides had hemolytic activity. Peptides were tested for hemolysis at a concentration range of 0. Absorbance at nm is hemoglobin specific. Subtracted absorbance at nm is nonspecific.
The steps for this hemolysis assay are depicted in fig. For any given AMP, the entire protocol described in this section was repeated in triplicate to determine hemolytic activity. These cells were later extracted using papain and used for cytotoxicity assays. Results in the form of percentage growth were reported. Here, percentage growth is defined as the growth of cells exposed to peptide relative to unexposed control cells, cultured under identical conditions.