Tachyplesin accumulates heterogeneously within clonal E. coli and P. aeruginosa populations.

(A) Tachyplesin-NBD accumulation in stationary phase E. coli BW25113 treated with increasing concentrations of tachyplesin-NBD for 60 min. Shaded regions show low (blue) and high (red) tachyplesin-NBD accumulation within an isogenic E. coli BW25113 population. Each histogram reports 10,000 recorded events and it is a representative example of accumulation data from three independent biological replicates (Figure S1D-J). (B) Tachyplesin-NBD accumulation in nine stationary phase E. coli clinical isolates treated with 46 μg mL-1 tachyplesin-NBD for 60 min. Accumulation data for E. coli BW25113 are reproduced from panel A. (C) Heatmap showing the presence of antimicrobial resistance genes and phylogenetic data of the clinical isolates. Corresponding gene products and the antimicrobials these genes confer resistance to are reported in Data Set S1. (D-F) Tachyplesin-NBD accumulation in P. aeruginosa, K. pneumoniae and S. aureus treated with 0 or 46 μg mL-1 tachyplesin-NBD for 60 min. Each histogram in each panel shows a representative example of accumulation data from three independent biological replicates. (G) Representative fluorescent images depicting low and high tachyplesin-NBD accumulators within an E. coli BW25113 population. Bacteria were continuously exposed to 46 μg mL-1 tachyplesin-NBD for 60 min in a microfluidic mother machine device. Blue stars and red diamonds indicate low and high accumulators, respectively. Scale bar: 3 μm. (H and I) Representative fluorescent images of individual low and high tachyplesin-NBD accumulators, respectively. Blue and red lines show a 2.2 μm-long cross-sectional line used for measuring fluorescence profile values in J and K, with the origin on the left side. (J and K) Normalised median (solid line), lower and upper quartiles (dotted lines) of fluorescence profile values of E. coli BW25113 cells plotted against the distance along the left side origin of a 2.2 μm-long straight line in low accumulators (J) and high accumulators (K), respectively. Phenotype assignment was further verified via propidium iodide staining (see Figure 2G). Inset: each dot represents the corresponding membrane to cell centre fluorescence ratio measured at the points indicated in H and I, dashed lines indicate the median and quartiles of each distribution. Statistical significance was assessed using an unpaired nonparametric Mann-Whitney U test with a two-tailed P-value and confidence level at 95%. ****: p<0.0001. Data was collected from three independent biological replicates.

Intracellular tachyplesin accumulation is essential for its antimicrobial efficacy.

(A and B) Fluorescence and side scatter values of individual E. coli treated with M9 at 37 °C for 60 min (A) and 46 μg mL-1 tachyplesin-NBD in M9 at 37 °C for 60 min (B). The black rectangles show the gates used to sort approximately one million untreated control cells, low and high tachyplesin-NBD accumulators for subsequent analysis. Data collected from four independent biological replicates. (C) Survival fraction of cells sorted through the untreated control, low and high tachyplesin-NBD accumulator gates presented in A and B. Bars and error bars represent the mean and standard deviation of data obtained from four biological replicates, each comprising three technical replicates. Statistical significance was assessed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test and confidence level at 95%. ****: p<0.0001, ns: p>0.05. (D-F) Representative microscopic images depicting brightfield (D), tachyplesin-NBD fluorescence (E), and propidium iodide (PI) fluorescence (F) after exposure to 46 μg mL -1 tachyplesin-NBD in M9 followed by a wash in M9 for 60 min, then 30 µM PI staining for 15 min at 37 °C in the microfluidic mother machine. Blue stars and red diamonds indicate low and high accumulators, respectively. Scale bar indicates 2.5 μm. (G) Correlation between tachyplesin-NBD and PI fluorescence of N = 371 individual E. coli cells collected from three independent biological replicates. The purple dashed line shows a nonlinear regression (semi-log) of the data (r2 = 0.70, p-value < 0.0001). (H) Correlation between the proportion of low accumulators as a percentage of the whole bacterial population measured via flow cytometry and survival fraction measured via colony-forming unit assays. Symbols and error bars represent the mean and standard deviation of three independent biological replicates, each comprising three technical replicates. Tachyplesin-NBD treatment concentration indicated by colour gradient. Some error bars are masked by the data points. The purple dashed line illustrates a linear regression of the data (r2 = 0.98, p-value < 0.0001).

Biological processes facilitating low tachyplesin accumulation.

(A and B) Log2 fold changes in transcript reads of genes in low and high tachyplesin-NBD accumulators (blue and red violin plots, respectively) relative to untreated stationary phase E. coli bacteria in cluster 2 (A) and cluster 4 (B). Each dot represents a single gene, dashed lines indicate the median and quartiles of each distribution. Dotted lines represent a log2 fold change of 0. Statistical significance was tested using a paired two-tailed Wilcoxon nonparametric test (due to non-normally distributed data) with a two-tailed p-value and confidence level at 95%. ****: p-value < 0.0001. The full list of genes belonging to each cluster are reported in Data Set S2 and the violin plots of log2 fold changes for all clusters are shown in Figure S9. Data was collected from four independent biological replicates then pooled for analysis. (C and D) Corresponding gene ontology enrichment plots of biological processes enriched in cluster 2 and 4. Each dot represents a biological process with its size indicating the number of genes associated with each process and the colour indicating the false discovery rate (FDR). The lines and their thickness represent the abundance of mutual genes present within the connected biological processes. The enrichment plot for cluster 5 is not shown as only one biological process, “response to biotic stimulus”, was enriched. Full details about each process are reported in Data Set S3. (E) Relative abundance of lipid classes (PG, phosphatidylglycerol; FA, fatty acids; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine) in untreated bacteria and bacteria treated with 46 µg mL-1 tachyplesin-NBD for 60 min. (F) Inferred transcription factors (TFs) activity, reported as normalised enrichment scores (NES), for the ten TFs with highest inferred activity in low or high accumulators (blue and red bars, respectively). Activity was inferred using Data Set S2 and a full list of TFs and the genes they regulate within each cluster is reported in Data Set S4.

Low accumulators respond to tachyplesin treatment by enhancing efflux.

(A) Distribution of tachyplesin-NBD accumulation in stationary phase E. coli over 240 min of treatment with 46 μg mL-1 tachyplesin-NBD in M9 at 37 °C. (B-D) Distribution of tachyplesin-NBD accumulation over 240 min after 15 min pretreatment in 46 μg mL-1 tachyplesin-NBD in M9 then washed and transferred into M9 (B), CCCP (50 μg mL-1) (C), or sertraline (30 μg mL-1) (D). Graphs are representative of three independent biological replicates and report 10,000 events. (E and F) Correlation of tachyplesin-NBD and PI fluorescence of individual stationary phase E. coli cells measured in the microfluidic mother machine after 60 min of 46 μg mL-1 tachyplesin-NBD treatment (N=108) (E) or cotreatment with 46 μg mL-1 tachyplesin-NBD and 30 μg mL-1 sertraline (N=108) (F). Purple dashed lines show nonlinear (semi-log) regressions (r2 = 0.68 and 0.38, respectively). (G) Survival fraction of stationary phase E. coli over 240 min treatment with tachyplesin (46 μg mL-1, magenta squares) or sertraline (30 μg mL-1, green circles), cotreatment with tachyplesin and sertraline (46 μg mL-1 and 30 μg mL-1, respectively, purple triangles), or incubation in M9 (black diamonds). Symbols and error bars indicate the mean and standard deviation of measurements performed in three biological replicates consisting of three technical replicates each. Some error bars are masked behind the symbols.