Targeted killing of pathogenic bacteria without harming beneficial members of host microbiota holds promise as a strategy to cure disease and limit both antimicrobial-related dysbiosis and development of antimicrobial resistance. We engineer toxins that are split by inteins and deliver them by conjugation into a mixed population of bacteria. Our toxin–intein antimicrobial is only activated in bacteria that harbor specific transcription factors. We apply our antimicrobial to specifically target and kill antibiotic-resistant Vibrio cholerae present in mixed populations. We find that 100% of antibiotic-resistant V. cholerae receiving the plasmid are killed. Escape mutants were extremely rare (10−6–10−8). We show that conjugation and specific killing of targeted bacteria occurs in the microbiota of zebrafish and crustacean larvae, which are natural hosts for Vibrio spp. Toxins split with inteins could form the basis of precision antimicrobials to target pathogens that are antibiotic resistant.
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The data, plasmids and strains generated for this study, that support our findings are available upon request to D. Mazel.
The Evolving Threat of Antimicrobial Resistance: Options for Action 1–119 (World Health Organization, 2014).
Davies, J. & Davies, D. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev. 74, 417–433 (2010).
Chikindas, M. L., Weeks, R., Drider, D., Chistyakov, V. A. & Dicks, L. M. Functions and emerging applications of bacteriocins. Curr. Opin. Biotech. 49, 23–28 (2018).
Bikard, D. et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).
Citorik, R. J., Mimee, M. & Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145 (2014).
Rodriguez-Pagan, I., Novick, R. P., Ross, H. F., Jiang, D. & Ram, G. Conversion of staphylococcal pathogenicity islands to CRISPR-carrying antibacterial agents that cure infections in mice. Nat. Biotechnol. 36, 971–976 (2018).
Lin, D. M., Koskella, B. & Lin, H. C. Phage therapy: an alternative to antibiotics in the age of multi-drug resistance. World J. Gastrointest. Pharmacol. Ther. 8, 162 (2017).
Jayaraman, P., Holowko, M. B., Yeoh, J. W., Lim, S. & Poh, C. L. Repurposing a two-component system-based biosensor for the killing of Vibrio cholerae. ACS Synth. Biol. 6, 1403–1415 (2017).
Lobato-Márquez, D. et al. Toxin-antitoxins and bacterial virulence. FEMS Microbiol. Rev. 40, 592–609 (2016).
Goeders, N. & Van Melderen, L. Toxin-antitoxin systems as multilevel interaction systems. Toxins 6, 304–324 (2013).
Ali, M., Nelson, A. R., Lopez, A. L. & Sack, D. A. Updated global burden of cholera in endemic countries. PLoS Negl. Trop. Dis. 9, 1–13 (2015).
Childers, B. M. & Klose, K. E. Regulation of virulence in Vibrio cholerae: the ToxR regulon. Future Microbiol. 2, 335–344 (2007).
Beaber, J. W., Hochhut, B. & Waldor, M. K. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427, 72–74 (2004).
Iqbal, N., Guérout, A. M., Krin, E., Le Roux, F. & Mazel, D. Comprehensive functional analysis of the 18 Vibrio cholerae N16961 toxin-antitoxin systems substantiates their role in stabilizing the superintegron. J. Bacteriol. 197, 2150–2159 (2015).
Guérout, A. M. et al. Characterization of the phd-doc and ccd toxin-antitoxin cassettes from Vibrio superintegrons. J. Bacteriol. 195, 2270–2283 (2013).
Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014).
Li, Y. Split-inteins and their bioapplications. Biotechnol. Lett. 37, 2121–2137 (2015).
Alford, S. C., O’Sullivan, C., Obst, J., Christie, J. & Howard, P. L. Conditional protein splicing of [small alpha]-sarcin in live cells. Mol. Biosyst. 10, 831–837 (2014).
Zettler, J., Schütz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914 (2009).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Crawford, J. A., Kaper, J. B. & DiRita, V. J. Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae. Mol. Microbiol. 29, 235–246 (1998).
Osorio, C. R. & Klose, K. E. A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among vibrio species. J. Bacteriol. 182, 526–528 (2000).
Lee, S. E. et al. Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA. J. Bacteriol. 182, 3405–3415 (2000).
Vezzulli, L., Pruzzo, C., Huq, A. & Colwell, R. R. Environmental reservoirs of Vibrio cholerae and their role in cholera. Env. Microbiol. Rep. 2, 27–33 (2010).
Runft, D. L. et al. Zebrafish as a natural host model for Vibrio cholerae colonization and transmission. Appl. Environ. Microbiol. 80, 1710–1717 (2014).
Austin, B., Austin, D., Sutherland, R., Thompson, F. & Swings, J. Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii. Environ. Microbiol. 7, 1488–1495 (2005).
Lennon, C. W. & Belfort, M. Inteins. Curr. Biol. 27, R204–R206 (2017).
Callahan, B. P., Topilina, N. I., Stanger, M. J., Van Roey, P. & Belfort, M. Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications. Nat. Struct. Mol. Biol. 18, 630–633 (2011).
Zhu, F. X. et al. Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors. Sci. China Life Sci. 56, 262–267 (2013).
Hacker, J. & Kaper, J. B. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641–679 (2000).
Main-Hester, K. L., Colpitts, K. M., Thomas, G. A., Fang, F. C. & Libby, S. J. Coordinate regulation of Salmonella pathogenicity island 1 (SPI1) and SPI4 in Salmonella enterica serovar typhimurium. Infect. Immun. 76, 1024–1035 (2008).
Loc-Carrillo, C. & Abedon, S. Pros and cons of phage therapy. Bacteriophage 1, 111–114 (2011).
Yosef, I., Manor, M., Kiro, R. & Qimron, U. Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl Acad. Sci. USA 112, 7267–7272 (2015).
Stirling, F. et al. Rational design of evolutionarily stable microbial kill switches. Mol. Cell 68, 686–696 (2017).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P(BAD) promoter. J. Bacteriol. 177, 4121–4130 (1995).
Bartolomé, B., Jubete, Y., Martínez, E. & de la Cruz, F. Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102, 75–78 (1991).
Poulin-Laprade, D. & Burrus, V. A γ Cro-like repressor is essential for the induction of conjugative transfer of SXT/R391 elements in response to DNA damage. J. Bacteriol. 197, 3822–3833 (2015).
Demarre, G. et al. A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains. Res. Microbiol. 156, 245–255 (2005).
Val, M. E., Skovgaard, O., Ducos-Galand, M., Bland, M. J. & Mazel, D. Genome engineering in Vibrio cholerae: a feasible approach to address biological issues. PLoS Genet. 8, e1002472 (2012).
Biskri, L., Bouvier, M., Guérout, A., Boisnard, S. & Mazel, D. Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities. J. Bacteriol. 187, 1740–1750 (2005).
We would like to thank E. Krin and M. Gugger for providing chromosome DNA for V. cholerae and V. fischeri, and N. punctiforme cells, respectively, and V. Burrus for V. cholerae O139. We thank G. Cambray and Z. Baharoglu for V. cholerae-GFP strain and RFP-containing plasmid, respectively. We thank S. Jin for her technical help. We thank also P. Escoll for assistance with microscopy, V. Briolat for providing us with the zebrafish and Artemias, A. Gomez-Losada for his help with the statistics treatment and S. Aguilar-Pierlé for helpful reading of the manuscript. We thank F. de la Cruz for his invaluable comments along the development of this work. This work was supported by the Institut Pasteur (to D.M. and J.-M.G.’s units), the Centre National de la Recherche Scientifique (grant no. CNRS-UMR 3525) (to D.M.), PLASWIRES 612146/FP7- FET-Proactive (to D.M.’s unit, A.R.-P.’s laboratory and for R.L.-I.’s salary), the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR−10-LABX-62-IBEID to D.M. and J.-M.G.’s units), Spanish project TIN2016-81079-R (AEI/FEDER, EU) and Comunidad de Madrid (cofunded by FES and FEDER, EU) S2017/BMD-3691 project ingeMICS-CM (to A.R.-P.), the Fondation pour la Recherche Médicale (grant no. DBF20160635736 to D.M. and DEQ20140329508 to J.-M.G.). J.B.-B. was the recipient of a long-term post-doctoral fellowship from the Federation of European Biochemical Societies.
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Toxicity comparison of single CDS toxin gene versus synthetic split toxin-intein fusion.
(a) Characteristics of plasmids pTox, pToxInt and pN. All constructions are derivative of pBAD43 a low copy number plasmid conferring spectinomycin (Sp) resistance. The pTox plasmid contains the ccdB toxin gene (red arrow) cloned under PBAD promoter, while in pToxInt ccdB is disrupted by the dnaE split intein (blue rectangles), forming an operon (N and C terminal genes fusion). As a control we used the non-toxic CcdB-DnaE-N fusion plasmid, the pN plasmid. Numbers below plasmids show the transformation rates into E. coli in repressible condition (glucose) for toxin or toxin-intein expression. (b) Growth curve in liquid media containing Sp and glucose, of 18 transformants clones from pTox (red), pToxInt (blue) or pN (gray) plasmids. No differences were found between growth rate of bacteria containing the non-toxic pN plasmid and pToxInt plasmid. However, bacteria containing pTox plasmid were not able to growth in repressible conditions for toxin expression (n=18 mean ± s.d.).
(a) Schematic representation of antitoxin-ccdA (pCcdA) and ccdB (pFW) expressing plasmids (left). When both plasmids are together in a bacterium, toxin-antitoxin complex allow bacteria to survive. Upon switch off of antitoxin expression ( + Glucose), the toxin is able to carry out its bactericidal or bacteriostatic effect. (b) V. cholerae O139 containing pCcdA and pPW plasmids grown in the presence of arabinose (allowing the antitoxin to be expressed) was diluted at OD = 0,5 (time 0h), arabinose was removed and glucose was added in order to switch off antitoxin expression for 4h at 37 °C. Viability at t = 0 and t = 4h was assessed by serial dilutions plated in drops on selective media + arabinose or + glucose. (c) Total bacteria (c.f.u./ml) able to grow on MH media + arabinose 0.2% at time 0h and 4h. After 4h upon exposure with the toxin, an important proportion of V. cholerae cells are not detected when the antitoxin is switched on (arabinose). * represents the proportion of mutants with CcdB inactivated, as they are able to grow in glucose conditions. This result indicates the bactericidal effect of CcdB toxin. Data numbers were calculated from four independent experiments (n = 4, mean ± s.d.).
(a) 3D-structure model from Phyre2 of each toxin. The protein sequence follows rainbow colors from blue (N-terminal) to red (C-terminal). Split sites are indicated by white arrows. (b) Amino acid sequences of the four toxins we split. These toxins are: CcdB from V. fischeri, and ParE2, HigB2 and RelE4 from V. cholerae. Structural properties in their amino acid sequences are highlighted as follows: blue represents beta sheet and green represents alpha helix. These properties are predicted using Phyre2. Split sites are indicated by a red arrow. For RelE4, we tested two sites (1 and 2) which were both functional.
Supplementary Figure 4 Toxins from different families are reconstituted after intein-mediated splicing.
Viability of E. coli DH5α bacteria containing different combination of plasmids with different toxins-intein fusions. Toxins and their corresponding families are indicated on the left. The split intein used is DNAE from Nostoc punctiforme. The left panel presents the viability assays using the native intein that carry out normal protein splicing; the right panel presents the viability assays using a N -terminal intein mutant (n*), in which residues CLS, which correspond to the catalytic Cys and the two following aminoacids from Nterminal domain were replaced by WER and splicing process does not take place (Cell. Mol. Life Sci., Volkmann, Mootz, 2013). Glucose represses the expression of the fusions and all strains grow. In presence of IPTG and arabinose (IPTG+Ara) bacteria containing both fusion plasmids (N+C) die. However bacteria containing only one half of the toxin-intein fusion (N or C) or the mutated version of N plasmid (n*) survive. Constructions and experimental set-up are described in the online methods. Experiments were repeated independently 3 times for the intact intein and twice for the inactivated version, with identical results.
a) Schematic representation of pU-BAD and pRS plasmids. pU-BAD plasmid contains the N terminal domain of CcdB-intein fusion under the promoter ompU which is activated by ToxRS. C terminal domain of CcdB-intein fusion is under the control of PBAD promoter. pRS plasmid contains toxRS genes from V. cholerae also under the control of PBAD promoter. In the presence of arabinose, the expression of C terminal and toxRS genes is induced and then, ToxRS will activate N terminal expression. (b) Growth test in solid media of E. coli DH5α bacteria containing different combination of pU-BAD or the empty vector (pBAD43, gray Ø) with pRS or its empty vector (pBAD30, pink Ø). In glucose conditions all bacteria survive. In arabinose, the presence of either single plasmids, pU-BAD or pRS, does not kill bacteria, while bacteria containing both pU-BAD and pRS plasmids die. Pictures are representative of three independent experiments (n = 3).
Supplementary Figure 6 Test of pU-BAD and of pPW, the genetic pathogenic-weapon, in V. cholerae serogroups O1 and O139.
(a) Growth test on solid media of V. cholerae serogroups O1 and O139, and of the O1 mutant lacking toxRS (ΔtoxRS) carrying pU-BAD plasmid. In presence of glucose all bacteria survive. In arabinose, only wild type serogroups of V. cholerae that contain toxRS die, but not the ΔtoxRS mutant. The colonies in arabinose for the two wild type V. cholerae strains illustrate the instability of pU-BAD plasmid in the presence of ToxRS. (b) Conjugation of the genetic weapon pPW into V. cholerae O1, O139 or the ΔtoxRS-mutant as recipients from the E. coli donor b3914. The ompU promoter activated specifically by ToxRS from V. cholerae is represented by a circled + pink-symbol. Selection of transconjugants was done on Sp containing media. Upon conjugation of pPW plasmid, V. cholerae O1 and O139 die, while the transconjugants of the ΔtoxRS mutant are fully viable. Pictures are representative of three independent experiments (n=3).
(a) Conjugation of pNctrl, pToxctrl or pPW plasmids into 1:1 mixed population of V. cholerae O1 (white) and E. coli MG1655 (blue) as recipients. Transconjugants were selected on appropriate antibiotic media containing arabinose and X-gal. The non-toxic pNctrl plasmid conjugates the mixed population, and pToxctrl plasmid kills both sub-populations. Upon conjugation of pPW plasmid only pathogenic V. cholerae O1 (white) die while E. coli MG1655 transconjugants are unaffected (blue). The ompU promoter activated specifically by ToxRS from V. cholerae is represented by a (+) pink-symbol. (b) pPW do not kill Salmonella typhimurium and Citrobacter rodentium. Transconjugants from pNctrl, pToxctrl or pPW plasmids conjugation were selected on Sp containing media. Transconjugants from control plasmids pNctrl and pToxctrl show that both bacterial strains are able to be conjugated in these conditions, and killed by the toxin-intein fusion. pPW transconjugants in these bacteria are fully viable, confirming that our system kills specifically only in the presence of ToxRS. Pictures are representative from three independent experiments.
Supplementary Figure 8 Design and test of PL promoter to control the ccdA antitoxin expression to prevent the CcdB killing activity.
(a) Schematic representation of pPLA plasmid which is the pToxInt plasmid (Supplementary Fig. 1) containing ccdA antitoxin under the control of the SXT PL promoter. (b) Growth test in solid media of E. coli lacking or containing SXT element chromosomally integrated (WT and SXT, respectively) transformed by pPLA. These strains were grown in the presence of glucose, then washed and re-suspended at OD = 0.5 in MH media containing glucose or arabinose, then spotted directly on glucose or arabinose containing media (0h) or after 1 h incubation at 37 °C with arabinose (1h). Pictures are representative from three independent experiments.
(a) Transconjugants selected in MH media with spectinomycin and X-gal. pNctrl plasmid containing pSC101 was used as control of conjugation. pNctrl and pPW plasmids containing R6K replication origin were able to replicate after conjugation only when E. coli λpir was used as recipient but not into V. cholerae O139. (b) Killing assay after conjugation in V. cholerae O139 of pPW-R6K, pFW-R6K and pNctrl-R6K non-replicative plasmids. As one cannot select for transconjugants, we measure the number of viable V. cholerae after conjugation with the 3 plasmids. Conjugation of the pNctrl-R6K non-replicative plasmid show that conjugation by itself do not affect V. cholerae viability, i.e. similar c.f.u./ml than for the not conjugated bacteria (V. cholerae). When conjugated with either pPW-R6K or pFW-R6K non-replicative weapons, the V. cholerae O139 population decrease by 2–3 fold indicating that even if not replicating both plasmids efficiently kill V. cholerae O139. One-way ANOVA with Dunnett’s Multiple Comparison Test was performed. pNctrl-R6K vs pPW-R6K, Mean Diff. = 2.383e + 008, q = 4.183, **P < 0.05, 95% CI of diff = (8.937e + 007 to 3.871e + 008). pNctrl-R6K vs pFW-R6K, Mean Diff. = 2.308e + 008, q = 4.227, **P < 0.05, 95% CI of diff = (9.187e + 007 to 3.896e+008). Data numbers were calculated from four independent experiments (n = 4, mean ± s.d).
Supplementary Figure 10 Localization of V. cholerae-GFP and E. coli-RFP in the two in vivo models, zebrafish (Danio rerio) and A.salina.
(a) Four-day-postfertilization zebrafish larvae were exposed to water containing V. cholerae-GFP and E. coli-RFP as described in methods. Infected and non-infected larvae were visualized by fluorescence microscopy using appropriate wavelength conditions enabling or not the visualization of GFP and RFP. Fluorescence was only detected in infected larvae and more precisely in the gut where both bacteria are co-localized. (b) Artemia salina stage nauplii were exposed to sterile PBS containing V. cholerae-GFP and E. coli-RFP as described in methods. Infected and non-infected nauplii were visualized by fluorescence microscopy using appropriate wavelength conditions enabling or not the visualization of GFP and RFP. Fluorescence was only detected in infected nauplii and more precisely in the gut where both bacteria are co-localized. Pictures are representative from three independent experiments.
Supplementary Figure 11 Analysis of zebrafish larvae microbiota and validation of the weapon in vivo.
(a) Analysis of the non-infected and infected with V. cholerae O139 zebrafish larvae’s microbiota. As a control, four-day post-fertilization zebrafish larvae were exposed to water containing 106 of β3914 bacteria carrying pNctrl or pFW plasmid for 24 h at 27 °C, and then microbiota from five larvae were plated in MH media. Blue bacteria on MH X-gal correspond to V. cholerae O139 as attested by their yellow color on TCBS Vibrio specific media. (b) Transconjugants selected from five larvae of zebrafish larvae after pNctrl or pFW conjugation treatment on MH media with Sp and X-gal. Blue colonies were only detected upon conjugation of pNctrl plasmid but not after pFW conjugation due to the specific killing of V. cholerae by this plasmid. Pictures are representative from the experiment with only V. cholerae O139 shown in Fig. 4a. (c) Transconjugants selected from the infection with pFW of 1:1 mixed population of V. cholerae O1 and O139, on MH media with Sp and X-gal and then replicated on TCBS plates to follow yellow development specific of Vibrio growth. Pictures are representative of the experiments using mix of Vibrio in Fig. 4a. Pictures from (a) and (c) are representative from two independent experiments (10 zebrafish larvaes) and from (b) are representative from three independent experiments (15 zebrafish larvaes).
Supplementary Figure 12 Analysis of A. salina stage nauplii microbiota and validation of the weapon efficacy in this model.
(a) Transconjugants selected from 225 ± 15 Artemia after infection with V. cholerae O139 and after treatment with β3914 bacteria carrying pNctrl or pFW conjugative plasmids, on MH media with Sp and X-gal. Blue colonies (V. cholerae O139) were only detected after conjugation with pNctrl plasmid but not after pFW conjugation, illustrating the specific killing of V. cholerae by pFW. Pictures are representative from the experiment with only V. cholerae O139 shown in Fig. 4b. (b) Transconjugants selected after infection with a mixed population of 1:1 V. cholerae O1 and O139, and treatment with β3914 bacteria carrying the pFW conjugative plasmid. Selection was done into MH media with Sp and X-gal and then, replicated on TCBS plates to detect the yellow color specific of Vibrio presence. As expected pFW transconjugants were only detected in V. cholerae O1 (white). Pictures are representative from the experiment with mix of Vibrios in Fig. 4b. Pictures are representative from four independent experiments (900 ± 15 nauplii of Artemia).
After conjugation of pABRW, in either E. coli SXT (its specific target) or E. coli MG1655 (not targeted) the number of surviving recipient bacteria (c.f.u./ml) was determined by plating on MH media without antibiotic and with arabinose (0,2%). One-sided t-test Mann Withney was performed. E. coli SXT vs E. coli MG1655. P value = 0.0143. *P < 0.05. Data numbers were calculated from four independent experiments (n = 4, mean ± s.d.).
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López-Igual, R., Bernal-Bayard, J., Rodríguez-Patón, A. et al. Engineered toxin–intein antimicrobials can selectively target and kill antibiotic-resistant bacteria in mixed populations. Nat Biotechnol 37, 755–760 (2019). https://doi.org/10.1038/s41587-019-0105-3
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