Journal: Nature Communications

Article Title: Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance

doi: 10.1038/s41467-019-12364-6

Figure Lengend Snippet: MIC and relative fitness of adapted lines after the laboratory evolution. a Relative resistance level in laboratory evolved E. coli K-12 BW2511 lines exposed to one of each 14 AMPs (blue) or 12 antibiotics (red), respectively (at least 9 parallel-evolved lines per drug). Altogether, lines exposed to AMPs ( N = 138) developed significantly lower resistance, than lines exposed to antibiotics ( N = 120) ( P < 0.0001, one-sided permutation test). The resistance levels reached were more heterogeneous across AMP treatments ( N = 14) than across antibiotic treatments ( N = 12) ( P = 0.03478 F -test). Each data point represents the MIC fold change of one of each parallel-evolved line. The mutD5 mutator strain exposed to TPII is marked by an asterisk (*). b Relative resistance level after laboratory evolution in clinical isolates under TPII or PXB stresses, respectively. Evolved lines exposed to TPII reached significantly lower resistance level than lines exposed to PXB (*** indicate the significant difference at least P -value = 1.65 × 10 –4 , two-sided Mann–Whitney test, N = 10 each group). Each data point represents the MIC fold change of one of each parallel-evolved line. c Relative fitness of 60 antibiotic-resistant and 38 AMP-resistant lines displaying at least twofold increments in resistance level to the drug indicated. Fitness was measured as the area under the growth curve in an antibacterial agent-free medium and was normalized to that of the wild-type (gray color). Throughout Fig. 1, boxplots show the median, first and third quartiles, with whiskers showing the 5th and 95th percentile. For AMP and antibiotic abbreviations, see Supplementary Tables – . Data in this figure are representative of at least two biological replicates. Source data are provided as a Source Data file

Article Snippet: Recombineering was performed in the electrocompetent E. coli K-12 BW25113 cells carrying pORTMAGE3 (Addgene plasmid ID: 72678). pORTMAGE3 was induced at 42 °C for 15 min to allow for efficient mutation-incorporation while avoiding off-target mutagenesis .

Techniques: MANN-WHITNEY

Journal: Nature Communications

Article Title: Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance

doi: 10.1038/s41467-019-12364-6

Figure Lengend Snippet: Resistance level correlate with AMPs’ physicochemical features. Each datapoint shows the average MIC-fold change in laboratory evolved E. coli K-12 BW2511 lines exposed to one of each 14 AMPs. a Fraction of polar amino acids and relative resistance level (Spearman’s rho = 0.58; p = 0.03, N = 14). b Fraction of positively charged and relative resistance level (Spearman’s rho = 0.62; p = 0.02, N = 14). c AMP hydropathicity and relative resistance level (Spearman’s rho = −0.73; p = 0.002, N = 14). For AMP properties, see Supplementary Data . Blue lines indicate the curve fitted using LOESS smoothing method in R. Source data are provided as a Source Data file

Article Snippet: Recombineering was performed in the electrocompetent E. coli K-12 BW25113 cells carrying pORTMAGE3 (Addgene plasmid ID: 72678). pORTMAGE3 was induced at 42 °C for 15 min to allow for efficient mutation-incorporation while avoiding off-target mutagenesis .

Techniques:

Journal: eLife

Article Title: Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

doi: 10.7554/eLife.46258

Figure Lengend Snippet: ( A ) Cartoon of LptD immunization campaigns with purified E. coli LptDE protein, LptD cyclic peptides, and linear peptides. 10 rounds of protein or peptide injections were performed. Clones were selected from hybridoma fusions. ( B ) Cartoon of targeted boost-and-sort strategy in which SD rats were initially primed with E. coli K-12 bacteria, followed by two boosts with the recombinant LptDE protein reconstituted in amphipol matrix. Cell hybridoma fusions were sorted with fluorescently-labeled LptDE to enrich LptDE + hybridomas. ( C ) Overview of the individual immunization campaigns. The total number of hybridoma clones isolated and binding to LptDE by ELISA were determined for each campaign. ELISA positive antibodies had a signal 3x above background. ELISA measurements were not determined (ND) for the hybridoma clones from the boost-and-sort approach.

Article Snippet: Bacterial immunization campaign immunized Sprague Dawley rats (Charles River Laboratories, Hollister, CA) with E. coli K-12 cells in PBS (10 7 –10 9 colony forming units via intravenous injection).

Techniques: Purification, Clone Assay, Recombinant, Labeling, Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

doi: 10.7554/eLife.46258

Figure Lengend Snippet: ( A ) LptD mAb campaign summary of ELISA + LptDE mAbs tested for surface binding on E. coli Δ waaD and E. coli K-12 by FACS assay. Antibodies were scored FACS + if the MFI was 2x above an isotype control. ( B ) Amino acids comprising the 13 ECLs of LptD are highlighted on the structure as spheres. The spheres are color-coded based on amino acid conservation between different sequence comparisons as indicated in the key. Structure is of Shigella flexneri LptDE with LptE removed rendered in PyMol (PDB 4Q35 ). ( C ) 134 FACS + and 233 FACS - antibodies were characterized for cross-reactive binding to purified LptDE from two closely-related Enterobacteriaceae species: Klebsiella pneumoniae ( K. pneum ) and Enterobacter cloacae ( E. cloacae ) by ELISA. ELISA positive antibodies had a signal 3x above background. ( D ) 52 FACS + α-LptD mAbs were characterized for epitope binning patterns using a high-throughput SPR-based method to determine pairwise mAb binding competition. Antibodies are color-coded based on ELISA cross-reactivity profiles as indicated in the key. Circle designates data obtained from both capture and detection antibodies. Square designates a single capture or detection data point. Characterization of all antibodies described in and those with green-outlined squares or circles are shown in .

Article Snippet: Bacterial immunization campaign immunized Sprague Dawley rats (Charles River Laboratories, Hollister, CA) with E. coli K-12 cells in PBS (10 7 –10 9 colony forming units via intravenous injection).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Purification, High Throughput Screening Assay

Journal: eLife

Article Title: Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

doi: 10.7554/eLife.46258

Figure Lengend Snippet: ( A ) Representative FACS traces for three ELISA + α-LptD mAbs, 27C2 (purple), 3D10 (blue), and 27C8 (red), with E. coli Δ waaD (top) and E. coli K-12 (bottom) cells. A non-binding isotype control mAb is shown in grey. 10,000 events were counted for each antibody and counts at each fluorescent intensity were plotted. ( B ) FACS analysis of a representative E. coli K-12 FACS + α-LptD mAb (27C8) for binding to E. coli Δ waaD strains expressing wild-type LptD and the 12 viable loop deletion strains. Loop 10 could not be assessed (see text). The mean fluorescent intensities (MFIs) are plotted. The red dashed line represents a descriptive MFI level that is 10% that of the WT control.

Article Snippet: Bacterial immunization campaign immunized Sprague Dawley rats (Charles River Laboratories, Hollister, CA) with E. coli K-12 cells in PBS (10 7 –10 9 colony forming units via intravenous injection).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Expressing

Journal: eLife

Article Title: Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

doi: 10.7554/eLife.46258

Figure Lengend Snippet: Representative Western blots of ( A ) log-phase E. coli K-12 wild-type liquid cultures and ( B ) E. coli Δ waaD cultures from overnight agar plates expressing each lptD loop mutant. Strains were grown in LB gentamicin with no arabinose. LptDΔL2 was severely growth delayed in E. coli K-12 and LptDΔL10 was not viable in E. coli Δ waaD so these could not be evaluated for protein expression. ( C ) Growth curves of lptD loop mutants expressed in a E. coli K-12 (left) and E. coli Δ waaD (right) strain background are shown. Strains were grown from a starting inoculum of OD 600 0.001 in LB gentamicin supplemented with arabinose and monitored for bacterial growth by measuring OD 600 over 15 hr in the presence of arabinose. Means and standard deviations for biological triplicates are plotted and comparisons of growth rates are shown in .

Article Snippet: Bacterial immunization campaign immunized Sprague Dawley rats (Charles River Laboratories, Hollister, CA) with E. coli K-12 cells in PBS (10 7 –10 9 colony forming units via intravenous injection).

Techniques: Western Blot, Expressing, Mutagenesis

Journal: eLife

Article Title: Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

doi: 10.7554/eLife.46258

Figure Lengend Snippet: Bacterial growth was measured (OD 600 ) for ( A ) E. coli Δ waaD and ( B ) E. coli K-12 after treatment with one representative plate of α-LptDE mAbs at 10 µg/mL for 4 hr. Black dashed line indicates media only control. Blue dashed line indicates 50% growth inhibition compared to no treatment.

Article Snippet: Bacterial immunization campaign immunized Sprague Dawley rats (Charles River Laboratories, Hollister, CA) with E. coli K-12 cells in PBS (10 7 –10 9 colony forming units via intravenous injection).

Techniques: Inhibition

Journal: Cell reports

Article Title: Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype

doi: 10.1016/j.celrep.2018.08.093

Figure Lengend Snippet: (A) E. coli rDNA operon organization. The prerRNA transcript is expressed from two tandem promoters (P 1 and P 2 ), encodes a 16S rRNA, one or more tRNA genes, a 23S rRNA, and a 5S rRNA and is terminated at two tandem terminators (t 1 and t 2 ). (B) Map of the E. coli chromosome indicating the location of all seven rDNA operons. The origin of replication is indicated by ‘‘Ori.’’ (C) 70S ribosome structure with a tRNA (red) in the peptidyl (P) site. Large subunit (50S) ribosomal proteins (blue), small subunit (30S) ribosomal proteins (tan), and rRNA (gray) are shown, and rRNA variant positions are rendered as spheres and colored by element. (D) 30S ribosomal subunit (solvent-side view) with major structural regions labeled, where rRNA variant positions are rendered as spheres and colored by element. PDB: 3R8T . (E and F) Polysome profiles ofwild-type E. coli grown in (E) complex or (F) minimal media. (G) The percent change of rDNA operons that are up-(green) or downregulated (red) in minimal media relative to complex media (p < 0.01). rDNA operons are listed on the x axis in order of their proximity to the origin of replication .

Article Snippet: E. coli K-12 MG1655 cells were purchased from ATCC (#700926).

Techniques: Variant Assay, Solvent, Labeling

Journal: Cell reports

Article Title: Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype

doi: 10.1016/j.celrep.2018.08.093

Figure Lengend Snippet:

Article Snippet: E. coli K-12 MG1655 cells were purchased from ATCC (#700926).

Techniques: Virus, Recombinant, Lysis, Sample Prep, SYBR Green Assay, Software