DOI: 10.1002/cbic.201500276
Highlights
Chemical Talking with Living Systems: Molecular Switches Steer Quorum Sensing in Bacteria Luca Schweighauser and Hermann A. Wegner*[a]
Most biological processes are controlled by changes on the molecular level. Therefore, treating diseases with small molecules has been highly successful in the past decades. However, controlling the pharmacokinetics of such chemical compounds is difficult, as their action cannot be turned on and off. In recent years molecular switches, which are able to change their structure following an external stimulus, have been promoted for steering the effect of a molecule after it has been administrated to a biological system.[1] Light as an external stimulus offers the great advantage of high spatial and temporal resolution, it is ideally nondestructive and, with a suitable wavelength, can be administered through tissue. There are a number of such photochromic compounds; one of the most prominent components of these is the azobenzene unit.[2] Azobenzene undergoes isomerization of the N=N double bond from the thermodynamically more stable E to the Z form upon irradiation with light. During this process, the molecule changes in length from ~ 9.0 to ~ 5.5 æ. In addition, azobenzenes are highly stable and show a very low fatigue during the switching process.[3] Due to the high potential of the azobenzene switch, intensive research in the past years has allowed the tuning of its properties such as stability of the Z isomers and the wavelength required for the isomerization. The general scheme for exploiting azobenzene as a switchable ligand to steer biological functions relies on the difference of the interaction with a specific receptor (Scheme 1). Such
Scheme 1. Controlling biological functions with the azobenzene scaffold.
functions range from controlling neuronal activity[4a] to bacterial adhesion.[4b] Moreover, a photoswitchable ligand for G protein-coupled adenosine receptors based on the azobenzene moiety has been designed and shown to act reversibly upon irradiation with different wavelengths.[5] The concept of “azo[a] L. Schweighauser, Prof. Dr. H. A. Wegner Institut fìr Organische Chemie, Justus-Liebig-Universitt Heinrich-Buff-Ring 58, 35392 Gießen (Germany) E-mail: [email protected]
ChemBioChem 2015, 16, 1709 – 1711
logization, the rational introduction of azobenzenes into drugs” has been also showcased in the modification of drugs such as fomocaine,[6a] fentanyl,[6b] propofol[6c] and antibacterial agents.[6d] Small molecules play an important role in communication between living systems.[7a] The entire communication between bacteria, known as the quorum sensing (QS) system relies on small molecules, autoinducers, to transfer information between individual bacteria so that they proliferate or build biofilms, etc.[7b] The reversible control of such autoinducers by an external stimulus, such as light, would open tremendous opportunities in bacterial engineering. Although Blackwell and co-workers suggested seven years ago that the incorporation of a molecular switch within an autoinducer would allow the QS to be steered, for example, by light,[8] it is only now that a collaborative effort by Driessen and Feringa with their teams has realized this vision in a proof-of-concept study.[9] They designed switchable variants of the azobenzene scaffold based on N-acyl homoserine lactones (AHLs),[10] which are naturally occurring autoinducers in the QS of a broad variety of bacteria, (Scheme 2). The three analogous presented in the study differ in the length of the tether between the switching unit and the lactone moiety as well as the substitution at the end of the azobenzene. First, the photochemical requirements of the switchable autoinducers were evaluated. Under irradiation all of them can be converted to the Z form as the preferred isomer. Whereas compound 2 shows only a moderate E:Z ratio of 1:2, 4 can be nearly quantitatively switched to the Z isomer. However, 2 exhibits the highest stability of the Z form in H2O at 30 and 37 8C. Compound 3 has the shortest half-life of only 2.5 h, but this is still sufficient for the activity assays (< 2 h). The ability of molecular switches 2–4 to control communication in bacteria was tested by steering the major QS regulatory network of Pseudomonas aeruginosa, the so called Las system.[7b] If the concentration of AHLs is below a certain threshold, the transcriptional activator LasR will be deactivated due to misfolding. With high bacteria density AHL levels will increase, and this allows LasR to fold correctly and activate the transcription of QS-controlled genes by binding to the responsive promoter regions on the bacterial genome. To test the effect of the switchable AHLs on Las QS, the biosensor Escherichia coli JM109 pSB1075 was treated with 2–4. Because of the presence of a luxCDABE-lasR promoter fusion, an increase in bioluminescence should be observed when the compounds activate the Las system. In the
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights presence of the native AHL 1 as well as the switchable AHLs 2 365 nm before the addition, expression of lasA was boosted and 3, a dose-dependent increase in bioluminescence was 18-fold. A control experiment with the P. aeruginosa DlasRI found (Figure 1); however, compound 4 showed no effect. If strain, which has no las receptor showed no effect of the analogues 2 and 3 were irradiated at 365 nm before incubaswitchable AHL, thus proving the specific up-regulation by Las tion, compound 2 caused a significant decrease in efficiency QS. and potency, whereas compound 3 had the opposite effect. After irradiation, the activity increased nearly fivefold. Compound 4 did not have any effect on QS, even after irradiation. The different activities of compounds 2–4 can be rationalized by the structural dissimilarities. While (E)-2 and (Z)-3 (with Scheme 2. Molecular design of a natural AHL, 1, and the switchable autoinducers 2–4 for QS. its more flexible tether) adopt a linear shape that allows a suitable interaction with the hydrophobic pocket, (Z)-2 and (E)-3 are more bent and block the necessary interaction with the receptor protein. The methoxy group makes compound 4 more polar, and this weakens the binding. The effects of 2 and 3 can be switched on and off reversibly. For 2, bioluminescence is high after irradiation with visible light and low in the presence of UV light. For 3 the opposite behaviour is observed (Figure 1). Under the testing conditions, the switchable AHLs were not stable. The authors attribute this to the instability of the lactone ring. However, it has been shown that glutathione, which is present in most cells, destroys the azo unit, and this might be Figure 1. Effect of the switchable AHLs on Las QS-controlled bioluminescence in the E. coli JM109 pSB1075 sensor an alternative explanation.[11] The Las QS system in P. aeru- strain. Reproduced from ref. [9], copyright: Royal Society of Chemistry, 2015. ginosa regulates a number of downstream processes, inter alia gene expression, such as that of the lasA gene encoding the protease LasA, which is responsible for bacteriolysis.[12] After being grown to a late exponential growth phase, the P. aeruginosa PA14 DlasI strain, which cannot produce Las QS AHLs, was treated with compound 3. lasA gene expression increased twofold after the addition of the nonirradiated 3 (Figure 2 A). When com- Figure 2. Las gene expression and pyocyanin production in P. aeruginosa controlled by a photoswitchable autopound 3 was irradiated at inducer. Reproduced from ref. [9], copyright: Royal Society of Chemistry, 2015. ChemBioChem 2015, 16, 1709 – 1711
www.chembiochem.org
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights Besides gene expression, QS also regulates phenotypes, such as biofilm formation, motility and toxin production. In P. aeruginosa pyocyanin, a toxin that harms competing bacteria and mammalian cells, is secreted under the control of QS. If the photoswitchable AHL 3 is added to the P. aeruginosa DlasI strain, only small amounts of pyocyanin are produced (Figure 2 B). However, if 3 was irradiated prior to administration, a substantial increase in the production was observed. These studies clearly demonstrate the power of molecular switches to steer biological functions. Driessen, Feringa and co-workers’ example is remarkable for being the first time QS, the complex communication system of bacteria, could be controlled simply by irradiation with light. So far, irradiation has only been done before the addition, as UV light would damage the bacteria. However, developments in the switching of azobenzene using IR wavelengths offer a way to overcome this challenge. Although the stability of switchable autoinducers and their efficiencies have to be improved, the proof-ofconcept will stimulate further research in this area, thus offering tremendous potential, for example, for bacterial engineering. Keywords: azobenzene · bacteria · molecular switches · photoswitches · quorum sensing [1] W. Szymanski, J. M. Beierle, H. A. V. Kistemaker, W. A. Velema, B. L. Feringa, Chem. Rev. 2013, 113, 6114 – 6178.
ChemBioChem 2015, 16, 1709 – 1711
www.chembiochem.org
[2] C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer, A. Heckel, Angew. Chem. Int. Ed. 2012, 51, 8446 – 8476; Angew. Chem. 2012, 124, 8572 – 8604. [3] A. A. Beharry, G. A. Woolley, Chem. Soc. Rev. 2011, 40, 4422 – 4437. [4] a) R. H. Kramer, D. L. Fortin, D. Trauner, Curr. Opin. Neurobiol. 2009, 19, 544 – 552; b) T. Weber, V. Chandrasekaran, I. Stamer, M. B. Thygesen, A. Terfort, T. K. Lindhorst, Angew. Chem. Int. Ed. 2014, 53, 14583 – 14586; Angew. Chem. 2014, 126, 14812 – 14815. [5] M. I. Bahamonde, J. Taura, S. Paoletta, A. A. Gakh, S. Chakraborty, J. Hernando, V. Fernndez-DueÇas, K. A. Jacobson, P. Gorostiza, F. Ciruela, Bioconjugate Chem. 2014, 25, 1847 – 1854. [6] a) M. Schoenberger, A. Damijonaitis, Z. Zhang, D. Nagel, D. Trauner, ACS Chem. Neurosci. 2014, 5, 514 – 518; b) M. Schçnberger, D. Trauner, Angew. Chem. Int. Ed. 2014, 53, 3264 – 3267; Angew. Chem. 2014, 126, 3329 – 3332; c) L. Yue, M. Pawlowski, S. S. Dellal, A. Xie, F. Feng, T. S. Otis, K. S. Bruzik, H. Qian, D. R. Pepperberg, Nat. Commun. 2012, 3, 1095; d) W. A. Velema, J. P. van der Berg, M. J. Hansen, W. Szymanski, A. J. M. Driessen, J. M. Arnold, Nat. Chem. 2013, 5, 924 – 928. [7] a) P. Williams, K. Winzer, W. C. Chan, M. Camara, Philos. Trans. R. Soc. London Ser. B 2007, 362, 1119 – 1134; b) M. B. Miller, B. L. Bassler, Annu. Rev. Microbiol. 2001, 55, 165 – 199. [8] G. D. Geske, J. C. O’Neill, D. M. Miller, R. J. Wezeman, M. E. Mattmann, Q. Lin, H. E. Blackwell, ChemBioChem 2008, 9, 389 – 400. [9] J. P. Van der Berg, W. A. Velema, W. Szymanski, A. J. M. Driessen, B. L. Feringa, Chem. Sci. 2015, 6, 3593 – 3598. [10] C. Fuqua, E. P. Greenberg, Nat. Rev. Mol. Cell Biol. 2002, 3, 685 – 695. [11] W. G. Levine, Drug Metab. Rev. 1991, 23, 253 – 309. [12] M. Schuster, E. P. Greenberg, Int. J. Med. Microbiol. 2006, 296, 73 – 81.
Manuscript received: June 2, 2015 Accepted article published: June 15, 2015 Final article published: July 14, 2015
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights
Chemical Talking with Living Systems: Molecular Switches Steer Quorum Sensing in Bacteria Luca Schweighauser and Hermann A. Wegner*[a]
Most biological processes are controlled by changes on the molecular level. Therefore, treating diseases with small molecules has been highly successful in the past decades. However, controlling the pharmacokinetics of such chemical compounds is difficult, as their action cannot be turned on and off. In recent years molecular switches, which are able to change their structure following an external stimulus, have been promoted for steering the effect of a molecule after it has been administrated to a biological system.[1] Light as an external stimulus offers the great advantage of high spatial and temporal resolution, it is ideally nondestructive and, with a suitable wavelength, can be administered through tissue. There are a number of such photochromic compounds; one of the most prominent components of these is the azobenzene unit.[2] Azobenzene undergoes isomerization of the N=N double bond from the thermodynamically more stable E to the Z form upon irradiation with light. During this process, the molecule changes in length from ~ 9.0 to ~ 5.5 æ. In addition, azobenzenes are highly stable and show a very low fatigue during the switching process.[3] Due to the high potential of the azobenzene switch, intensive research in the past years has allowed the tuning of its properties such as stability of the Z isomers and the wavelength required for the isomerization. The general scheme for exploiting azobenzene as a switchable ligand to steer biological functions relies on the difference of the interaction with a specific receptor (Scheme 1). Such
Scheme 1. Controlling biological functions with the azobenzene scaffold.
functions range from controlling neuronal activity[4a] to bacterial adhesion.[4b] Moreover, a photoswitchable ligand for G protein-coupled adenosine receptors based on the azobenzene moiety has been designed and shown to act reversibly upon irradiation with different wavelengths.[5] The concept of “azo[a] L. Schweighauser, Prof. Dr. H. A. Wegner Institut fìr Organische Chemie, Justus-Liebig-Universitt Heinrich-Buff-Ring 58, 35392 Gießen (Germany) E-mail: [email protected]
ChemBioChem 2015, 16, 1709 – 1711
logization, the rational introduction of azobenzenes into drugs” has been also showcased in the modification of drugs such as fomocaine,[6a] fentanyl,[6b] propofol[6c] and antibacterial agents.[6d] Small molecules play an important role in communication between living systems.[7a] The entire communication between bacteria, known as the quorum sensing (QS) system relies on small molecules, autoinducers, to transfer information between individual bacteria so that they proliferate or build biofilms, etc.[7b] The reversible control of such autoinducers by an external stimulus, such as light, would open tremendous opportunities in bacterial engineering. Although Blackwell and co-workers suggested seven years ago that the incorporation of a molecular switch within an autoinducer would allow the QS to be steered, for example, by light,[8] it is only now that a collaborative effort by Driessen and Feringa with their teams has realized this vision in a proof-of-concept study.[9] They designed switchable variants of the azobenzene scaffold based on N-acyl homoserine lactones (AHLs),[10] which are naturally occurring autoinducers in the QS of a broad variety of bacteria, (Scheme 2). The three analogous presented in the study differ in the length of the tether between the switching unit and the lactone moiety as well as the substitution at the end of the azobenzene. First, the photochemical requirements of the switchable autoinducers were evaluated. Under irradiation all of them can be converted to the Z form as the preferred isomer. Whereas compound 2 shows only a moderate E:Z ratio of 1:2, 4 can be nearly quantitatively switched to the Z isomer. However, 2 exhibits the highest stability of the Z form in H2O at 30 and 37 8C. Compound 3 has the shortest half-life of only 2.5 h, but this is still sufficient for the activity assays (< 2 h). The ability of molecular switches 2–4 to control communication in bacteria was tested by steering the major QS regulatory network of Pseudomonas aeruginosa, the so called Las system.[7b] If the concentration of AHLs is below a certain threshold, the transcriptional activator LasR will be deactivated due to misfolding. With high bacteria density AHL levels will increase, and this allows LasR to fold correctly and activate the transcription of QS-controlled genes by binding to the responsive promoter regions on the bacterial genome. To test the effect of the switchable AHLs on Las QS, the biosensor Escherichia coli JM109 pSB1075 was treated with 2–4. Because of the presence of a luxCDABE-lasR promoter fusion, an increase in bioluminescence should be observed when the compounds activate the Las system. In the
1709
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights presence of the native AHL 1 as well as the switchable AHLs 2 365 nm before the addition, expression of lasA was boosted and 3, a dose-dependent increase in bioluminescence was 18-fold. A control experiment with the P. aeruginosa DlasRI found (Figure 1); however, compound 4 showed no effect. If strain, which has no las receptor showed no effect of the analogues 2 and 3 were irradiated at 365 nm before incubaswitchable AHL, thus proving the specific up-regulation by Las tion, compound 2 caused a significant decrease in efficiency QS. and potency, whereas compound 3 had the opposite effect. After irradiation, the activity increased nearly fivefold. Compound 4 did not have any effect on QS, even after irradiation. The different activities of compounds 2–4 can be rationalized by the structural dissimilarities. While (E)-2 and (Z)-3 (with Scheme 2. Molecular design of a natural AHL, 1, and the switchable autoinducers 2–4 for QS. its more flexible tether) adopt a linear shape that allows a suitable interaction with the hydrophobic pocket, (Z)-2 and (E)-3 are more bent and block the necessary interaction with the receptor protein. The methoxy group makes compound 4 more polar, and this weakens the binding. The effects of 2 and 3 can be switched on and off reversibly. For 2, bioluminescence is high after irradiation with visible light and low in the presence of UV light. For 3 the opposite behaviour is observed (Figure 1). Under the testing conditions, the switchable AHLs were not stable. The authors attribute this to the instability of the lactone ring. However, it has been shown that glutathione, which is present in most cells, destroys the azo unit, and this might be Figure 1. Effect of the switchable AHLs on Las QS-controlled bioluminescence in the E. coli JM109 pSB1075 sensor an alternative explanation.[11] The Las QS system in P. aeru- strain. Reproduced from ref. [9], copyright: Royal Society of Chemistry, 2015. ginosa regulates a number of downstream processes, inter alia gene expression, such as that of the lasA gene encoding the protease LasA, which is responsible for bacteriolysis.[12] After being grown to a late exponential growth phase, the P. aeruginosa PA14 DlasI strain, which cannot produce Las QS AHLs, was treated with compound 3. lasA gene expression increased twofold after the addition of the nonirradiated 3 (Figure 2 A). When com- Figure 2. Las gene expression and pyocyanin production in P. aeruginosa controlled by a photoswitchable autopound 3 was irradiated at inducer. Reproduced from ref. [9], copyright: Royal Society of Chemistry, 2015. ChemBioChem 2015, 16, 1709 – 1711
www.chembiochem.org
1710
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Highlights Besides gene expression, QS also regulates phenotypes, such as biofilm formation, motility and toxin production. In P. aeruginosa pyocyanin, a toxin that harms competing bacteria and mammalian cells, is secreted under the control of QS. If the photoswitchable AHL 3 is added to the P. aeruginosa DlasI strain, only small amounts of pyocyanin are produced (Figure 2 B). However, if 3 was irradiated prior to administration, a substantial increase in the production was observed. These studies clearly demonstrate the power of molecular switches to steer biological functions. Driessen, Feringa and co-workers’ example is remarkable for being the first time QS, the complex communication system of bacteria, could be controlled simply by irradiation with light. So far, irradiation has only been done before the addition, as UV light would damage the bacteria. However, developments in the switching of azobenzene using IR wavelengths offer a way to overcome this challenge. Although the stability of switchable autoinducers and their efficiencies have to be improved, the proof-ofconcept will stimulate further research in this area, thus offering tremendous potential, for example, for bacterial engineering. Keywords: azobenzene · bacteria · molecular switches · photoswitches · quorum sensing [1] W. Szymanski, J. M. Beierle, H. A. V. Kistemaker, W. A. Velema, B. L. Feringa, Chem. Rev. 2013, 113, 6114 – 6178.
ChemBioChem 2015, 16, 1709 – 1711
www.chembiochem.org
[2] C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer, A. Heckel, Angew. Chem. Int. Ed. 2012, 51, 8446 – 8476; Angew. Chem. 2012, 124, 8572 – 8604. [3] A. A. Beharry, G. A. Woolley, Chem. Soc. Rev. 2011, 40, 4422 – 4437. [4] a) R. H. Kramer, D. L. Fortin, D. Trauner, Curr. Opin. Neurobiol. 2009, 19, 544 – 552; b) T. Weber, V. Chandrasekaran, I. Stamer, M. B. Thygesen, A. Terfort, T. K. Lindhorst, Angew. Chem. Int. Ed. 2014, 53, 14583 – 14586; Angew. Chem. 2014, 126, 14812 – 14815. [5] M. I. Bahamonde, J. Taura, S. Paoletta, A. A. Gakh, S. Chakraborty, J. Hernando, V. Fernndez-DueÇas, K. A. Jacobson, P. Gorostiza, F. Ciruela, Bioconjugate Chem. 2014, 25, 1847 – 1854. [6] a) M. Schoenberger, A. Damijonaitis, Z. Zhang, D. Nagel, D. Trauner, ACS Chem. Neurosci. 2014, 5, 514 – 518; b) M. Schçnberger, D. Trauner, Angew. Chem. Int. Ed. 2014, 53, 3264 – 3267; Angew. Chem. 2014, 126, 3329 – 3332; c) L. Yue, M. Pawlowski, S. S. Dellal, A. Xie, F. Feng, T. S. Otis, K. S. Bruzik, H. Qian, D. R. Pepperberg, Nat. Commun. 2012, 3, 1095; d) W. A. Velema, J. P. van der Berg, M. J. Hansen, W. Szymanski, A. J. M. Driessen, J. M. Arnold, Nat. Chem. 2013, 5, 924 – 928. [7] a) P. Williams, K. Winzer, W. C. Chan, M. Camara, Philos. Trans. R. Soc. London Ser. B 2007, 362, 1119 – 1134; b) M. B. Miller, B. L. Bassler, Annu. Rev. Microbiol. 2001, 55, 165 – 199. [8] G. D. Geske, J. C. O’Neill, D. M. Miller, R. J. Wezeman, M. E. Mattmann, Q. Lin, H. E. Blackwell, ChemBioChem 2008, 9, 389 – 400. [9] J. P. Van der Berg, W. A. Velema, W. Szymanski, A. J. M. Driessen, B. L. Feringa, Chem. Sci. 2015, 6, 3593 – 3598. [10] C. Fuqua, E. P. Greenberg, Nat. Rev. Mol. Cell Biol. 2002, 3, 685 – 695. [11] W. G. Levine, Drug Metab. Rev. 1991, 23, 253 – 309. [12] M. Schuster, E. P. Greenberg, Int. J. Med. Microbiol. 2006, 296, 73 – 81.
Manuscript received: June 2, 2015 Accepted article published: June 15, 2015 Final article published: July 14, 2015
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
