Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria

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Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells Pengbo Zhang, Huan Lu, Hui Chen, Jiangyan Zhang, Libing Liu,* Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China S Supporting Information *

ABSTRACT: Bacteria quorum sensing (QS) has attracted significant interest for understanding cell−cell communication and regulating biological functions. In this work, we demonstrate that water-soluble cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacteria QS system and prolong the time duration of QS signal molecules (autoinducer-2 (AI-2)) production. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to understand cell−cell communication and regulate biological functions, such as the production of signaling molecules, antibiotics, other microbial metabolites, and even virulence. ince its first discovery in the marine luminous bacteria Vibrio f ischeri (V. f ischeri) and Vibrio harveyi (V. harveyi), quorum sensing (QS) has attracted significant research interest.1−4 QS is defined as a cell-density dependent behavior in which bacteria mediate their cell density by sensing the concentration of small diffusible molecules, called autoinducers (AIs).5 Before bacteria enter stationary phase, the extracellular concentration of AIs, produced and secreted by the bacteria, is in proportion to cell density. Once the concentration of AIs reach a threshold, more AIs can bind with specific protein and regulate downstream gene expression which can trigger corresponding responses in their behaviors, such as bioluminescence,6 infection,7 sporulation,8 biofilm formation,9 virulence production,10 swarming motility,11 and so on. Also, AIs can help bacteria to optimize their growth condition and survive in the adverse environment.12 Up to date, many studies have been focused on how to inhibit QS in order to control bacterial infection. Piletska et al. have designed polymers with the ability to incorporate QS signal molecules of the bacteria V. f ischeri, resulting in attenuation of bioluminescence.13 Alexander and co-workers have prepared a series of polymers with different functional groups which can bind bacteria and/or QS signal molecules; these polymers are able to interfere with their QS system.14,15 However, using QS signal molecules to probe and manipulate the QS system should have significant importance for growing bacteria with a slow growth rate or inducing other microbial metabolites production. Conjugated polymers (CPs) have delocalized electronic structures which exhibit unique electronic and optical properties. In recent years, water-soluble conjugated polymers have

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attracted much attention because of their biological applications.16−20 Due to the collective amplification behaviors, trace detection of biomacromolecules (nucleic acids and proteins) and fluorescence imaging in vivo have been successfully accomplished using these water-soluble conjugated polymers.21−26 The current state of the art in the field focuses on their applications in highly sensitive diagnostics and photodynamic therapy (PDT); however, fewer studies have been demonstrated to utilize them in bacteria communication and QS signaling pathway. Dendronized polymers contain central linear polymeric cores and repeatedly dendronized groups which to some extent can improve the fluorescent quantum efficiency and prevent the self-aggregation of the polymer in aqueous solution. Dendrimers with terminal amino or quaternary ammonium moieties can provide polyvalent interactions with bacteria and help to form tight bacteria− polymer agglomerates.27,28 Herein, we demonstrate that watersoluble conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. Upon coating some bacteria in the aggregate, PFP-G2 has the ability to induce a bacterial QS system with prolonged time duration of QS signal molecules production. Those prolonged QS signal molecules can continuously regulate specific gene expression, resulting in decreased antimicrobial susceptibility. Received: October 16, 2015 Accepted: February 25, 2016

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DOI: 10.1021/acs.analchem.5b03920 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Thus, we provide a useful tool for understanding cell−cell communication and regulating biological functions. In our study, Escherichia coli (E. coli) MG1655 is chosen as the bacteria to interact with conjugated polymers. E. coli is a species of Gram-negative bacteria naturally present in human and animal bodies, which has a negatively charged membrane.29,30 The syntheses of the cationic dendronized poly(fluorene)s with positively charged amine groups on the exterior (PFP-G2) is reported previously.31 The proposed principle of the interactions of CPs with bacteria to form QS and the detection of QS signal molecules are illustrated in Scheme 1. Upon adding PFP-G2 to the culture medium Scheme 1. Schematic Representation of PFP-G2 for Aggregation of E. coli and Detection of QS Signal Molecules

Figure 1. (a) CLSM images of E. coli incubated in the absence and presence of PFP-G2. The fluorescence of PFP-G2 is highlighted in blue. Left, fluorescence image; middle, phase contrast bright-field image; right, overlapped image. (b) ζ potentials of E. coli before and after incubating with PFP-G2. Measurements were performed in water at 25 °C.

are also used to further investigate the interactions between PFP-G2 and E. coli. Upon addition of PFP-G2, ζ potentials of E. coli have a positive shift from −39.40 ± 3.01 mV to −26.8 ± 2.10 mV (Figure 1b), indicating that PFP-G2 successfully bind to the outer membranes of E. coli, while no obvious change of ζ potentials is observed for E. coli in the absence and presence of PFP-G2 after the mixture was washed by a solution of 500 mM NaCl. These observations prove that the binding of PFP-G2 to E. coli is dominated by electrostatic interactions, indeed supporting the mechanism presented in Scheme 1. We then investigated the effect of bacteria aggregation on the E. coli QS system using the V. harveyi strain as a reporter by bioluminescence. In the initial study, the characteristic QS behavior of V. harveyi is investigated. As shown in Figure 2a, in the first phase (Decay phase), the bioluminescence decreases as V. harveyi is diluted into fresh medium which has lower concentrations of AI-2 as compared to the preculture medium. At a critical cell density which corresponds to the detection threshold of AI-2, the bioluminescence of V. harveyi increases exponentially (Enhancement phase). Between the two phases, there is a time point of 3.5 h referred to the minimum luminescence of V. harveyi. We then prepare a series of cell-free culture fluids derived from E. coli only (Figure 2b,c) and E. coli incubated with PFP-G2 (Figure 2d,e) for 1−8 h. The addition of cell-free culture fluids causes V. harveyi to maintain a high level of light output after being incubated for 3.5 h (Figure 2b,e), while growth curves of V. harveyi in these conditions are very similar (Figure S1). The increased light output is due to V. harveyi in response to the presence of AI-2 produced by E. coli in cell-free culture fluids. To get more insights into the effect of PFP-G2 on the QS system of E. coli, the intensity of luminescence at 3.5 h in Figure 2b,d is recorded. As shown in Figure 2f, in the absence of PFP-G2, the AI-2 activity is produced maximally in midexponential phase and degraded when the bacteria enter stationary phase. While in the presence

solution of E. coli, electrostatic interactions bring them together to form PFP-G2/E. coli aggregates. After incubation for a period of time, those E. coli buried within the aggregates either can not obtain enough nutrition from culture medium solution or can not grow due to the confined space, also QS signal molecules produced by those bacteria are not able to diffuse away, resulting in increased QS signal molecules production. As the only QS signal molecule that functions in both Gramnegative and Gram-positive bacterial communication, autoinducer-2 (AI-2) is used as the model QS signal molecule. V. harveyi (BB170), a bioluminescent bacterium that produces light only in response to AI-2, is used as the reporter strain. The increased light output of V. harveyi is due to the presence of AI2 in the supernatant prepared from E. coli with PFP-G2. However, in the absence of PFP-G2, E. coli can not form aggregates. The concentration of AI-2 in the supernatant is lower than that in the presence of PFP-G2. Then, fewer AI-2 can return into V. harveyi and regulate downstream gene expression, resulting in decreased light output. We first proved that PFP-G2 exhibited good biological compatibility that could be used to interact with E. coli (Table S1). The conjugated backbones of PFP-G2 retain their optical properties, which serve to study the mechanism by visualizing their interactions with bacteria under confocal laser scanning microscopy (CLSM). As shown in Figure 1a, PFP-G2 and E. coli cells are aggregated to agglomerates; meanwhile, E. coli cells and the blue fluorescence of PFP-G2 are clearly overlapped, while control experiments show that E. coli without PFP-G2 does not aggregate in LB medium. Zeta (ζ) potentials B

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Our results have shown that AI-2 is not produced by the onset of stationary phase, which means the QS system does not function during stationary phase. While upon adding PFP-G2 to the culture medium solution of E. coli, the activity of AI-2 can last for a longer time after the bacteria enter stationary phase. In order to figure out the effect of prolonged activity of AI-2 on antimicrobial susceptibility, we measured the viability of E. coli after incubation with PFP-G2 for 5 and 6 h against ampicillin (AMP). After addition of AMP, the survival rate gradually declines whether PFP-G2 is added or not (Figure 3).

Figure 3. Survival rate of E. coli incubated with 100 μM PFP-G2 for 5 h (a) and 6 h (b) against 5 mg/mL AMP.

After incubation for 5 h, the concentration of AI-2 is similar between E. coli and E. coli with PFP-G2 (Figure 2f). Thus, the survival rate is almost the same after adding AMP. While after incubation for 6 h, E. coli with PFP-G2, which has a prolonged AI-2 production, has a higher survival rate, indicative of decreased antimicrobial susceptibility. Also, different concentrations of AMP are used to further measure the antimicrobial susceptibility of E. coli (Figure S2). The viability difference between E. coli incubated in the absence and presence of PFPG2 for 6 h becomes larger by increasing the concentration of AMP. These results suggest that the prolonged AI-2 production induced by PFP-G2 decreases the antimicrobial susceptibility of E. coli. We then investigate the effect of PFP-G2 on biofilm formation of E. coli using the crystal violet staining method.32 The results show that E. coli with PFP-G2 produces 55.54 ± 12.03% more biofilm than E. coli only (Table S2), which indicates that AI-2 induced by PFP-G2 stimulates biofilm formation in E. coli. In summary, the results herein demonstrate that cationic dendronized poly(fluorene)s with positively charged amine groups on the exterior can provide polyvalent interactions with bacteria and prolong the time duration of QS signal molecules production. The conjugated backbones retain their optical properties in dendronized polymers, which serve to study the mechanism by visualizing their interactions with E. coli under fluorescence microscopy. The CLSM and ζ potential measurements show that the binding of PFP-G2 to E. coli is dominated by electrostatic interactions. Upon adding PFP-G2 to the culture medium solution of E. coli, the activity of AI-2 can last for a longer time after the bacteria enter stationary phase. The prolonged AI-2 can return into bacteria, bind with specific protein, and regulate downstream gene expression. As a result, E. coli show a higher survival rate against AMP, indicative of decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to regulate biological functions, such as the production of signaling molecules, antibiotics, other microbial metabolites, and even virulence. As the production of AI-2 has been prolonged, other bacteria, which can interact with AI-2 in their surrounding environment, may undergo QS

Figure 2. (a) Luminescence spectra of V. harveyi. Luminescence spectra of V. harveyi in response to cell-free culture fluids extracted from E. coli incubated with 0 (b, c) and 100 μM (d, e) PFP-G2 for 1− 8 h. (f) Differences of luminescence intensity of V. harveyi at 3.5 h in response to cell-free culture fluids extracted from E. coli incubated with 0 and 100 μM PFP-G2 for 1−8 h. (g) OD600 of E. coli incubated with 0 and 100 μM PFP-G2 for 3−8 h.

of PFP-G2, the activity of AI-2 can last for at least 8 h which means PFP-G2 has the ability to prolong the time duration of AI-2 production. At the same time, the viability of E. coli in the presence of PFP-G2 is also monitored by measuring the absorbance at 600 nm (OD600) at a 1 h interval. As shown in Figure 2g, PFP-G2 influences the growth of E. coli to some degree. This lack of growth to some extent has an impact on luminescence intensity of V. harveyi, which explains why AI-2 production of E. coli with PFP-G2 is lower than that without PFP-G2 during 1−5 h of incubation. While after 6−8 h of incubation, AI-2 production of E. coli with PFP-G2 is increased, much higher than that without PFP-G2. The reason may be due to those E. coli buried within the aggregates either can not obtain enough nutrition from culture medium solution or can not grow due to the limited space; also, QS signal molecules produced by those bacteria are not able to diffuse away, resulting in increased AI-2 production. It is supposed that slow diffusion of AI-2 from the aggregates enable bacteria to detect a higher concentration of AI-2 rapidly, so E. coli incubated with PFP-G2 enter stationary phase at a lower value of OD600. These results confirm that PFP-G2, which can interact with E. coli to form PFP-G2/E. coli aggregates, has the ability to induce the bacterial QS system and extend the time duration of AI-2 production. C

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(15) Lui, L. T.; Xue, X.; Sui, C.; Brown, A.; Pritchard, D. I.; Halliday, N.; Winzer, K.; Howdle, S. M.; Trillo, F. F.; Krasnogor, N.; Alexander, C. Nat. Chem. 2013, 5, 1058. (16) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339. (17) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467. (18) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168. (19) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater. Chem. 2005, 15, 2648. (20) Feng, F.; He, F.; An, L.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2008, 20, 2959. (21) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 10954. (22) Nilsson, K. P. R.; Inganäs, O. Nat. Mater. 2003, 2, 419. (23) Abérem, M. B.; Najari, A.; Ho, H. A.; Gravel, J. F.; Nobert, P.; Boudreau, D.; Leclerc, M. Adv. Mater. 2006, 18, 2703. (24) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 7505. (25) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343. (26) Feng, F.; Tang, Y.; He, F.; Yu, M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2007, 19, 3490. (27) Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329. (28) Ortega, P.; Copa-Patiňo, J. L.; Muňoz-Fernandez, M. A.; Soliveri, J.; Gomez, R.; de la Mata, F. J. Org. Biomol. Chem. 2008, 6, 3264. (29) Nikaido, H.; Vaara, M. Microbiol. Rev. 1985, 49, 1. (30) Helander, I. M.; Alakomi, H. L.; Latva-Kala, K.; MattilaSandholm, T.; Pol, I.; Smid, E. J.; Gorris, L. G. M; von Wright, A. J. Agric. Food Chem. 1998, 46, 3590. (31) Yu, M.; Liu, L.; Wang, S. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 7462. (32) Gonzalez Barrios, A. F.; Zuo, R.; Hashimoto, Y.; Yang, L.; Bentley, W. E.; Wood, T. K. J. Bacteriol. 2006, 188, 305.

behavior without growing to high density. The system can also be used to initiate the QS system of some bacteria that can not grow to high density or the growth rate is slow.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03920. Experimental details including materials and methods, bioluminescence and growth of V. harveyi, preparation of cell-free culture fluids, detection of AI-2 using V. harveyi bioassay, confocal laser scanning microscopy (CLSM) characterization, Zeta potential measurements, drugresistance experiments, biological compatibility of PFPG2, biofilm formation assay, Figures S1 and S2, and Tables S1 and S2. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86-10-62636680. *E-mail: [email protected]. Fax: +86-10-62636680. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Major Research Plan of China (No. 2013CB932800) and the National Natural Science Foundation of China (Nos. TRR61, 21373243).

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REFERENCES

(1) Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165. (2) Hooshangi, S.; Bentley, W. E. Curr. Opin. Biotechnol. 2008, 19, 550. (3) Diggle, S. P.; Gardner, A.; West, S. A.; Griffin, A. S. Philos. Trans. R. Soc., B 2007, 362, 1241. (4) Geske, G. D.; O’ Neill, J. C.; Blackwell, H. E. Chem. Soc. Rev. 2008, 37, 1432. (5) Camilli, A.; Bassler, B. L. Science 2006, 311, 1113. (6) Surette, M. G.; Bassler, B. L. Mol. Microbiol. 1999, 31, 585. (7) Kong, K.-F.; Vuong, C.; Otto, M. Int. J. Med. Microbiol. 2006, 296, 133. (8) Bischofs, I. B.; Hug, J. A.; Liu, A. W.; Wolf, D. M.; Arkin, A. P. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 6459. (9) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (10) Miller, M. B.; Skorupski, K.; Lenz, D. H.; Taylor, R. K.; Bassler, B. L. Cell 2002, 110, 303. (11) Daniels, R.; Reynaert, S.; Hoekstra, H.; Verreth, C.; Janssens, J.; Braeken, K.; Fauvart, M.; Beullens, S.; Heusdens, C.; Lambrichts, I.; De Vos, D. E.; Vanderleyden, J.; Vermant, J.; Michiels, J. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 14965. (12) Williams, P.; Winzer, K.; Chan, W. C.; Camara, M. Philos. Trans. R. Soc., B 2007, 362, 1119. (13) Piletska, E. V.; Stavroulakis, G.; Karim, K.; Whitcombe, M. J.; Chianella, I.; Sharma, A.; Eboigbodin, K. E.; Robinson, G. K.; Piletsky, S. A. Biomacromolecules 2010, 11, 975. (14) Xue, X.; Pasparakis, G.; Halliday, N.; Winzer, K.; Howdle, S. M.; Cramphorn, C. J.; Cameron, N. R.; Gardner, P. M.; Davis, B. G.; Trillo, F. F.; Alexander, C. Angew. Chem., Int. Ed. 2011, 50, 9852. D

DOI: 10.1021/acs.analchem.5b03920 Anal. Chem. XXXX, XXX, XXX−XXX