Multiplexed bacteriocin synthesis to combat and prevent antimicrobial resistance

Abstract

Bacteriocins are underexplored yet promising candidates to combat antimicrobial resistance (AMR) and enable targeted therapy due to their natural origin, abundance and narrow spectrum of activity. In this study, we used a collection of engineered DNA devices and cell-free gene expression (CFE) to rapidly produce combinations (cocktails) of bacteriocins comprising both linear and circular proteins. Other cocktails were designed to target a specific bacterial species by leveraging insights into bacteriocin pathways for cell envelope penetration. These tailored combinations eradicated bacteria effectively while preventing resistance development. The synthesis of bacteriocins was optimized by using continuous exchange CFE, reengineering DNA parts, and adjusting conditions for disulfide bond formation. Also, we illustrate the efficacy of these bacteriocin mixtures against various multidrug-resistant human pathogens and highlight their potential through in vivo testing in the animal model Galleria mellonella. Our bacteriocin cocktail expression and test platform underscores the potential of bacteriocins for innovative treatments against multidrug-resistant infections.

Fig. 1. Bacteriocin in vitro genetic expression with the PARAGEN collection.

Bacteriocin expression devices are engineered for CFE using synthetic biology approaches, such as abstraction, standardization, modularization, and optimization. Genes coding for bacteriocin peptides and proteins are searched in bacterial genome databases and published research. Genetic parts coding for coding for either linear or circular bacteriocins are optimized in silico for codon usage and synthesized de novo together with standardized genetic parts, such as the T7 RNA polymerase promoter (P_T7), ribosome binding site (RBS), and transcription terminators (T₇ and T_rrnB). Only the coding sequence for the mature peptide is integrated in the genetic module, while for circular peptides, bacteriocin genes are fused with intein fragments for circularization. The bacteriocin expression devices are cloned into multi-copy vectors that can be transformed in E. coli for pDNA replication. A lac operator (lac_op) is included to repress leaky expression while plasmids are carried in E. coli and, thus, avoid toxicity by bacteriocin expression. Synthesis takes 2–3 h in vitro with conventional CFE kits. Thereafter, bacteriocins are tested on high-density bacterial cell lawns in their respective standard growth media and conditions.

Fig. 2. Rational designed and cell-free expressed bacteriocin cocktail.

A Bacteriocins pathways in E. coli cell envelope. Tol and Ton-dependent bacteriocin pathways are highlighted in orange and blue, respectively, as well as producer cells and bacteriocins targeting those pathways. Different colicins and microcins are distinguished with the Col and Mcc prefixes, respectively, while post-translationally modified bacteriocins are indicated with an asterisk (*). B Activity of different bacteriocins against E. coli BW25113. Halo of activity as seen by the naked eye (left) and under a stereo microscope to detail colonies appearing in the halos (right). C Growth (OD₆₀₀) of E. coli BW25113 in liquid cultures supplemented with single and co-expressed bacteriocins. D Viability (CFU ml⁻¹) of cells in cultures (from C) after 72 h. E CFU counts of E. coli BW25113 in liquid cultures supplemented with single and co-expressed bacteriocins 1250-fold diluted. Lines in (C and E) and bars in (D) represent the average of three biological replicates $\pm$ SD. Dots in (D) show the values of replicates.

Fig. 3. Optimization of PARAGEN sequences increase bacteriocin activity.

A Gene expression devices for CFE of GFP with and without optimization (gfp_opt, green box, and gfp, doted box, respectively). Measurement (RFU) of CFE of GFP devices. B, C Activity of ColE1 (B) and MccV (C) from CFE with and without optimization as seen against a top law of E. coli BW25113 and after measuring the MDIP. B Impact on ColE1 activity with the original PARAGEN device (+lacO + colE1), after removing either the lac operator (−lacO + colE1), after optimizing the codon usage of a bacteriocin gene (+lacO + colE1_opt) or in combining both optimization strategies (−lacO + colE1_opt). C Activity of MccV with or without optimization in presence or absence of supplements for disulfide bond formation (DSB) against a top law of E. coli BW25113, and minimum inhibitory dilution as seen on plates (MIDP) of CFE MccV optimized without and with a DSB supplement. D Activity on plates and liquid cultures of single and cocktail CFE of ColE1 and MccV with a DSB supplement. Bars (in A–C) and lines (D) are representative of the average value of three technical replicates (A) and the activity on three biological replicates, $\pm$ SD (in B–D). Dots in (A–C) show the values of replicates.

Fig. 4. CECFE scaling up and de-multiplexing bacteriocins in cocktail.

A Diagram of synthesis of the ColM + SalE1B cocktail with CEFCE using the RTS ProteoMaster Instrument (Roche). The feeding chamber (FC) contains substrates and energy cofactors, while the reaction chamber (RC) holds the pDNA, and transcription and translation machinery. Reactions are shaken (180 rpm) to improve diffusion of substrates into RC and of byproducts of protein synthesis out to FC. B MIDP of cocktail preparations using PURE CFE (PCFE) and CECFE reactions. C The activity of each bacteriocin in the cocktail was distinguished following separation by molecular weight with Tris-Tricine SDS-page and test of activity on a top lawn of E. coli BW25113. The blue and orange arrows indicate the predicted ColM and SalE1B bands, respectively. The uncropped image is included in Supplementary Information file. D LC-MS/MS proteomic analysis of the ColM and SalE1B cocktails produced using either PURE CFE or CECFE, confirming the high abundance of both bacteriocins in both cocktails. Protein ranking of all proteins detected is based on the intensity-based absolute quantification (iBAQ). A lower rank is correlated with a higher abundance. The estimated concentration of ColM and SalE1B, given between brackets, is calculated based on the known added concentration of trypsin (0.01 mg/ml). Bars in (B) represent the average and dots the value of three biological replicates $\pm$ SD.

Fig. 5. Diluted bacteriocin cocktail is not toxic and improves survival of Galleria mellonella after bacterial infection.

A, B Survival (A) and health (B) scores measurements on the animal model G. mellonella individuals after injection of different dilutions (1:8, 1:100, and 1:1000) of the ColM + SalE1B cocktail (C + S) produced by CECFE. C Survival score measurements on G. mellonella after infection with antibiotic multi-resistant E. coli strains (141, 302,425, 10037, 10270, 10276-2, 10500, 10611). Blue line in graph for treatment with isolate 10276-2 is superimposed on green one. n = 10 per treatment, ns not significantly different (p $\ge$ 0.05), * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical differences in survival rates were calculated using the Log-rank and Wilcoxon tests, with a significance level of p < 0.05.

Fig. 6. Cell-free co-expression of bacteriocins from PARAGEN targeting Gram-positive and -negative bacteria.

A Bacteriocin gene expression devices from the PARAGEN collection optimized for CFE of either EntL50A, SalE1B, or GarML. B Bacteriocin activity tests on E. coli DH10B and P. pentosaceus CWBI-B29. Cocktails of EntL50A + SalE1B and GarML + SalE1B were synthesized using PURE CFE (PurExpress, NEB).

Fig. 7. Co-expression and bacteriocin activity in combinations with three different DNA molecules.

A GFP fluorescence (RFU) throughout time in single and combined CFE. Lines represent the average value of three technical replicates and error bars ($\pm$ SD). B Bacteriocin activity tests on E. coli DH10B and P. pentosaceus CWBI-B29. C ColM + SalE1B + MccL bacteriocin cocktail activity tests on E. coli BW23115 mutants insensitive to two of the three bacteriocins. A–C All proteins were synthesized using PURE CFE (PurExpress, NEB).