University, Yoto, Utsunomiya, Tochigi , JAPAN

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1 Inhibition of Antimicrobial Prodigiosin Production in Serratia marcescens Using Cyclodextrin-Immobilized Microgel Beads Prepared by a Non-Contact Jet Dispensing System Eri Nasuno 1, a, Chigusa Okano 2, b, Yuriko Takayama 3, c, Izumi Harano 1, d, Ken-ichi Iimura 1, e, Norihiro Kato 1, f * 1 Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University, Yoto, Utsunomiya, Tochigi , JAPAN 2 Creative Department for Innovation, Collaboration Center for Research and Development, Utsunomiya University, Yoto, Utsunomiya, Tochigi , JAPAN 3 Department of Innovation Systems Engineering, Graduate School of Engineering, Utsunomiya University, Yoto, Utsunomiya, Tochigi , JAPAN a e-nasuno@cc.utsunomiya-u.ac.jp, b okanoc@cc.utsunomiya-u.ac.jp, c dt157106@cc.utsunomiya-u.ac.jp, d mt166333@cc.utsunomiya-u.ac.jp, e emlak@cc.utsunomiya-u.ac.jp, f katon@cc.utsunomiya-u.ac.jp Keywords: Cyclodextrin, Quorum sensing, Cell-cell communication, N-acylhomoserine lactone, Bacterial Infection Abstract. Some gram-negative bacteria possess N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) as one of the cell-to-cell communication systems for regulation of various cell functions. Increase in the AHL concentration above its threshold leads to simultaneous activation of target gene expression in each cell. The AHL can diffuse in and out of the cells through the cell membrane. Artificial reduction of the AHL concentration can inhibit the AHL complex formation with its receptor inside the cell. In this study, the AHL that diffused out into the culture broth was captured on α-cyclodextrin (CD), which possesses a hydrophobic cavity and a hydrophilic shell. The α-cd-immobilized calcium alginate gel beads, which were prepared using a non-contact jet dispenser, were immersed in the culture medium during the cell growth of Serratia marcescens AS-1, which was used as the model opportunistic human pathogen. The trapping of AHLs on immobilized α-cds due to hydrophobic interaction of the AHL acyl-chain led to an effective inhibition of AHL-mediated prodigiosin production to approximately 2% in the presence of 27 wt% gel beads in the culture broth. The results of our study suggest that CD has high potential to regulate QS involved in matter production, biofilm formation, and virulence factor expression. Introduction Quorum sensing (QS) is one of the bacterial cell-to-cell signaling systems that regulate various cell functions including bioluminescence [1], biofilm formation [2], and matter production [3]. These functions are possible due to the synchronization of the transcriptional activation of the QS-target genes [4]. A signaling molecule on QS is accumulated around the cells accompanied by increase in the cell density. When the cell number reaches a quorum, the N-acylhomoserine lactone (AHL) concentration increases enough to form a stable complex with its receptor protein. Then the complex interacts with the QS-target gene for the transcriptional activation. In QS for gram-negative bacteria, AHLs possessing various lengths of acyl chains are produced to regulate the QS-related operon [5]. In this study, we attempted to inhibit the AHL-mediated QS in Serratia marcescens by engineered reduction of the AHL concentration. The AHL-mediated QS system was first studied in marine bacterium Vibrio fischeri, which possesses a bioluminescence gene cluster consisting of eight lux genes [6, 7]. S. marcescens produces a transcriptional regulator SpnR as a LuxR family protein, which is the homolog protein of N-(3-oxohexanoyl)-L-homoserine lactone (3oxoC6HSL) receptor in V. fischeri (Fig. 1). The

2 formation of the SpnR complex with the AHL can trigger the beginning of the sequential QS process generally when the AHL concentration increases above the threshold due to cell multiplication. In the previous studies, we demonstrated the negative regulation of SpnR for the spn operon, which is related to the production of AHL-synthase and SpnR itself [8]. Specific interaction of SpnR with a promoter spn box was also proved in vitro by using a quartz crystal microbalance (QCM) analysis [9]. Fig. 1 A schematic illustration of quorum sensing in Serratia marcescens AS-1. To inhibit the bacterial QS systems by reducing the concentration of free AHL in the culture, inclusion complex formation between AHL and α-cyclodextrin (α-cd) was utilized in this study. The hydrophobic cavity of the cyclic glucopyranose was enabled to form a complex with the acyl chain of AHL via hydrophobic interaction in the culture [10-12]. Since AHLs trapped in the CDs become inactive, the control of gene expression via AHLs is blocked. Alginate was selected as the CD-immobilized matrix because it is stable in culture; it has been often used for the immobilization of microorganisms, owing to its safety and easy preparation. A non-contact jet dispensing system was successfully applied to prepare the sub-millimeter-sized microgel beads. Tiny droplets dispensing a viscous sodium alginate solution were shot into the calcium chloride solution. The resultant droplets immediately form hydrogel by chelating Ca 2+ ions to the guluronic acid domain of the alginate. The objectives of this study were to prepare α-cd-immobilized calcium alginate hydrogel beads using a non-contact jet dispenser and to demonstrate the inhibitory activity of the immobilized CDs in AHL-mediated QS of a model opportunistic human pathogen. This QS inhibitory technique is expected to enable the artificial control of bacterial metabolism such as pathogenicity and biofilm formation. Materials and Methods Materials. α-cd was kindly supplied by Cyclochem (Japan). Sodium alginate ( mpa s at 1 wt%) was purchased from Wako Pure Chemical. All chemicals were used without further purification. Immobilization of α-cd on Alginate. Aqueous solutions of sodium alginate and α-cd were mixed to adjust the concentrations as 3 and 10 wt%, respectively. To immobilize the α-cd, 50% glutaraldehyde aqueous solution was mixed at 60 C and stirred for 3 h. To improve the solubility of alginate in water, the mixture was autoclaved (121 C, 2 bar) for 15 min. The solution was filtered through a 0.45-μm mixed cellulose ester membrane and the resultant pre-gel solution was applied to a non-contact jet dispenser. To determine the amount of immobilized α-cd, the sample solution was dialyzed in water. The α-cd concentration in the outer solution, which contains the remaining α-cd after the glutaraldehyde reaction, was determined by high performance liquid chromatography (HPLC) equipped with a refractive index detector (RID-6A, Shimadzu). Size-exclusion chromatography was performed to separate α-cd and other impurities using an OH-possessing silica gel column (Shim-pack Diol-150, φ mm).

3 Preparation of Microgel Beads. The syringe filled with the pre-gel solution was set to non-contact jet dispenser for high viscosity (Aero Jet MJET-A, Musashi Engineering) equipped with an air pulse controller (MJET-3-CTR, Musashi Engineering). The pre-gel solution was dispensed by air pulse (0.132 MPa) and then shot into a gelling solution containing 0.5 wt% CaCl 2. To avoid clogging of the nozzle, pre-filtration of the pre-gel solution was required. The distance from the nozzle head to the liquid surface was set to 15 cm. The obtained gel beads were immersed in the gelling solution to complete the Ca 2+ ion penetration for 2 h, followed by washing with distilled water. The size distribution of the gel beads was determined by stereomicroscope measurement for 100 gel beads (M165C, Leica). Frequency distribution / number mm Diameter / mm Fig. 2 Size distribution of α-cd immobilized gel beads prepared using a non-contact jet dispenser. Inhibitory tests on QS in S. marcescens AS-1. The effects of exogenously added CD-immobilized gel beads on QS inhibition were studied with S. marcescens AS-1, which produces N-hexanoylhomoserine lactone (C6HSL) and 3oxoC6HSL as the signals on QS. The AS-1 cells were pre-cultured in Luria-Bertani (LB) medium at 25 C. After inoculation (1%) to fresh LB medium, the main culture was performed at 25 C for 15 h with or without the desired amount of CD-immobilized gel beads. As the index of activation and inhibition on QS, the amount of AHL-induced prodigiosin production was determined. The turbidity of the culture (OD 600 ) was measured as the index of cell density. After centrifugation of the culture broth, prodigiosin was extracted from the cells by re-suspension in acidified ethanol, followed by vigorous vortexing [13]. The absorbance at 534 nm (A 534 ) for the supernatant of the extracted solution was measured using an ultraviolet-visible spectrophotometer (V-630, JASCO). The prodigiosin production per cell unit was calculated as A 534 /OD 600. The A 534 /OD 600 values were normalized by control value without any additives during the cell growth. Results and Discussion Characterization of CD-Immobilized Gel Beads Prepared Using a Non-Contact Jet Dispenser. The immobilized amount of α-cd was determined as 41 mmol/g-alginate by HPLC analysis. Microscopic measurement suggested that the mode diameter of the gel beads is 500 m (Fig. 2), which does not depend on the immobilization of α-cd. The viscous alginate solution was dispensed to tiny droplets using a non-contact jet dispenser driven by an air compressor. The preparation rate of the gel beads could be controlled by the interval of the air pulse. We successfully prepared the gel beads at 70,000 shots/h. To avoid the fusion of gel beads due to collision, stirring of gelling solution was required. By applying the dispensing technique for the viscous solution, sub-millimeter-sized calcium alginate gel beads are stably prepared. Millimeter-sized beads were known to be easily prepared by adding sodium alginate solution to the gelling solution containing Ca 2+ ions. However,

4 the formation of smaller droplets from such viscous pre-gel solutions requires an air-compressed mechanical distribution of the solution. Inhibitory Effects of Immobilized α-cd on QS in S. marcescens. S. marcescens AS-1 was selected as a model opportunistic human pathogen that possesses the AHL-mediated QS [14]. After the AHL concentration increases above a threshold, C6HSL complex with its receptor SpnR becomes stable and then interacts with pig promoter to activate the expression of the pig clusters. The color of the culture broth turned red after reaching the stationary phase because the red pigment prodigiosin was accumulated inside the cell. Fig. 3 Inhibitory effects of α-cd immobilized in calcium alginate gel beads on prodigiosin production in S. marcescens AS-1 (25 C, 15 h). (a) Calcium alginate gel beads without α-cd. (b) Calcium alginate gel beads with α-cd (41 mmol/g-alginate). Left-pointing arrows indicate the axis on the bar graphs and the right-pointing arrows show the axis of the line graphs. In this study, 11, 20, or 27 wt% gel beads as the wet mass were immersed in LB medium at the beginning of the main culture. Prodigiosin was extracted from the cells after 15-h cell growth according to the standard method [13]. As compared with the control without any gel beads, added gel beads without α-cd had minimal effects on QS-dependent prodigiosin production (Fig. 3a). No growth inhibition was observed by the addition of gel beads. However, prodigiosin production was considerably reduced in the presence of calcium alginate gel beads immobilized with α-cd. Relative prodigiosin production in the presence of 20 and 27 wt% α-cd-immobilized gel beads was approximately 7 and 2%, respectively (Fig. 3b). Effective interaction between the immobilized CD and C6HSL was previously demonstrated by analyses of QCM [15] and 1 H-NMR [10]. The results of our study showed that the hydrophobic acyl chain of the AHL can be included into the hydrophobic cavity of the α- or β-cd in aqueous media due to the hydrophobic interaction. Such encapsulation of the QS signal is responsible for the maintenance of the inactive state of QS because the apparent concentration of the QS signal is too low to activate the expression of the target genes. In this study, we demonstrated a reproducible preparation of sub-millimeter-sized calcium alginate gel beads by using an air-compressed jet dispenser and their application in the engineered regulation of QS. Immobilized CD is the trusted carrier for the uptake of QS signals from the culture. Artificial control of the AHL concentration is one of the effective strategies to control the group behavior of bacteria, which possess the gene expression system dependent on the cell-to-cell signaling.

5 Conclusions The uniform-sized microgel beads immobilized with α-cd were successfully prepared using a non-contact jet dispensing system. Adding more than 20 wt% (w/v) of the α-cd immobilized gel beads effectively interrupted the prodigiosin production regulated by QS in S. marcescens AS-1. This was done by maintaining the concentration of free AHLs produced by S. marcescens AS-1 below a certain threshold by trapping into the immobilized α-cd in the culture. The effective inhibition of AHL-mediated QS systems could be proposed as one of the effective techniques for control of bacterial pathogenicity in the medical field as well as biofouling in wastewater treatment systems. Acknowledgments We thank M. Kaneko and Y. Tsunematsu for their technical assistance. References [1] J. Slock, D. vanriet, D. Kolibachuk, E. P. Greenberg, Critical regions of the Vibrio fischeri LuxR protein defined by mutational analysis, J. Bacteriol. 172(7) (1990) [2] D. G. Davies, M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, E. P. Greenberg, The involvement of cell-to-cell signals in the development of a bacterial biofilm, Sci. 280(5361) (1998) [3] M. Schuster, D. J. Sexton, S. P. Diggle, E. P. Greenberg, Acyl-homoserine lactone quorum sensing: From evolution to application, Annu. Rev. Microbiol. 67 (2013) [4] B. L. Bassler, R. Losick, Bacterially speaking, Cell 125(2) (2006) [5] T. Bjarnsholt, M. Givskov, Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens, Phil. Trans. R. Soc. B 362(1483) (2007) [6] N. A. Whitehead, A. M. L. Barnard, H. Slater, N. J. L. Simpson, G. P. C. Salmond, Quorum sensing in gram-negative bacteria, FEMS Microbiol. Rev. 25(4) (2001) [7] E. P. Greenberg, Quorum sensing in gram-negative bacteria, ASM News 63 (1997) [8] Y. Takayama, E. Nasuno, K. Iimura, T. Morohoshi, N. Kato, Quartz crystal microbalance analyses of SpnR binding constants as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens AS-1, Chem. Lett. 44(7) (2015) [9] Y. Takayama, N. Kato, In vitro analysis of essential binding sites on the promoter of the Serratia marcescens spn operon with the quorum-sensing receptor SpnR, Biotechnol. Bioeng. 113(11) (2016) [10] T. Morohoshi, K. Tokita, S. Ito, Y. Saito, S. Maeda, N. Kato, T. Ikeda, Inhibition of quorum sensing in gram-negative bacteria by alkylamine-modified cyclodextrins, J. Biosci. Bioeng. 116(2) (2013) [11] N. Kato, T. Tanaka, S. Nakagawa, T. Morohoshi, K. Hiratani, T. Ikeda, Control of virulence factor expression in opportunistic pathogens using cyclodextrin immobilized gel, J. Incl. Phenom. Macrocycl. Chem. 57(1) (2007) [12] C. Okano, E. Nasuno, K. Iimura, N. Kato, Cyclodextrin-immobilized microspheres for uptake of the quorum-sensing signaling molecule N-acylhomoserine lactone, J. Appl. Poly. Sci. 133(12) (2016) [13] H. Slater, M. Crow, L. Everson, G. P. C. Salmond, Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and -independent pathways, Mol. Microbiol. 47 (2003)

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