Quorum Sensing Disruption in Vibrio harveyi Bacteria by Clay

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Clay Catalyzed Disruption of Quorum Sensing in Vibrio harveyi Bacteria Sajo Naik, Jonathon Scholin, San Ching, Fang Chi, and Marc Herpfer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03918 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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Journal of Agricultural and Food Chemistry

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Quorum Sensing Disruption in Vibrio harveyi

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Bacteria by Clay Materials

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Sajo P. Naik, * Jonathon Scholin, San Ching, Fang Chi, and Marc

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Herpfer

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The Oil-Dri Innovation Center

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777 Forest Edge Road

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Vernon Hills, IL 60061-3197

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Corresponding Author Sajo P. Naik The Oil-Dri Innovation Center 777 Forest Edge Road Vernon Hills, IL 60061-3197 [email protected] Phone: +1- 847-537-7543

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ACS Paragon Plus Environment

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ABSTRACT

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This work describes the use of clay minerals as catalysts for the degradation of quorum sensing

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molecule, N-(3-Oxooctanoyl)-DL-homoserine lactone. Certain clay minerals due to their surface

32

properties and porosity can catalytically degrade the quorum sensing molecule into smaller

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fragments. The disruption of quorum sensing by clay in a growing Gram-negative Vibrio harveyi

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bacteria culture was also studied by monitoring luminescence and population density of the

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bacteria, wherein quenching of bacterial quorum sensing activity was observed by means of

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luminescence reduction. The results of this study show that food grade clays can be used as

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biocatalysts in disrupting bacterial activity in various media.

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KEYWORDS

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Clay, Biocatalysis, Transformations, N- (3-Oxooctanoyl)-DL-homoserine, quorum sensing

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1. Introduction

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Quorum sensing is a cell-to-cell communicating system employed by bacteria to regulate

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bacterial activity in response to various stimuli. Quorum sensing was first reported in the study of

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a marine bacteria Vibrio fischeri which exhibited bioluminescence upon bacteria reaching a high

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population density [1]. Typically, bacteria biosynthesize distinctive chemicals including N-

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acylhomoserine lactones (AHLs) as quorum sensing signals; specifically, AHLs, such as N-(3-

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Oxooctanoyl)-DL-homoserine lactone are used by some Gram-negative bacteria in quorum

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sensing signaling. The biochemical production and effectiveness of AHLs depend primarily on

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the activity of Lux and LuxR protein families, respectively, which together with the quorum

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sensing molecules, constitute the overall quorum sensing signaling system. After AHLs are

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produced by Lux enzymes, they diffuse across bacterial membranes and accumulate in the

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surroundings until critical local concentrations are reached [2]. At a given threshold

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concentration, the AHL binds to LuxR response regulator, forming a complex that activates gene

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expression. Some bacterial features controlled through quorum sensing include bacteria

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population, antibiotic generation, bioluminescence, nitrogen-fixing gene regulation, Ti plasmid

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conjugal transfer, virulent gene expression, pigment generation, bacterial swarming, and biofilm

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formation [3].

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Quorum quenching is a process of disrupting the bacterial quorum system. Modulation of Lux,

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LuxR and deactivation of quorum quenching molecules are some ways of accomplishing quorum

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quenching. There exist several reports on the use of naturally occurring or artificially synthesized

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antagonists as possible quorum quenchers [4,7]. Ideally, quorum sensing quenchers, inhibitors,

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or regulators should not interfere with the normal physiological functions of bacterial cells or not

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prompt bacteria to develop resistance. Although several different methods of quorum quenching

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are reported, to the best of our knowledge there does not exist any biocatalytic ways involving

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solid acid catalysts such as clays to disrupt quorum sensing in bacteria.

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Clay minerals are hydrous aluminum phyllosilicates, which may contain variable

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amounts of iron, magnesium, alkali metals, alkaline earths and other cations. Clay minerals exist

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in nature but must be further processed or purified such that their chemical or physical properties


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required in various applications may be brought to the fore. Clays have a very interesting history

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as solid acid catalysts. Acid-treated clays were used as catalysts in petroleum refining in the early

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1930s [8-9]. However, their applicability was limited due to their lack of thermal stability under

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high temperature fluidized catalytic cracking (FCC) conditions. Consequently, clays were

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replaced by more stable synthetic catalysts, e.g. silica-alumina, zeolites, etc. in the petrochemical

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industry. Besides catalysis clay minerals have several other applications as adsorbents, feed

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additives, fillers, carriers, etc. [8-13]. In several animal health products, clay minerals are used as

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feed additives as they are non-toxic, have high porosity and presence of active sites that can bind

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a range of fungal and microbial toxins to improve the health of the animal [10-13]. In the animal

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feed and health industry versatility of clays to perform more than one physiological functions is

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desired.

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Herein, through in vitro studies we demonstrate that clays can adsorb and catalyze

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bacterial quorum sensing molecules to break them into smaller fragments in an aqueous system.

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We used N-(3 Oxooctanoyl)-DL-homoserine lactone, an AHL type quorum sensing molecule as

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a model quorum sensing molecule for this in vitro study. We followed up the in vitro study by

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directly monitoring the quorum quenching in Vibrio harveyi bacteria in the presence of clays

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under simulated bacterial growth conditions. Bioluminescence and bacteria population were the

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parameters employed to gauge the quorum sensing activity of the system. Clay minerals

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functioned as adsorption/catalytic antagonists in that the AHLs responsible for quorum sensing

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activity in some bacteria were either separated out by adsorption or were catalytically broken

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down by the active site in clay surface.

Reducing the concentration of quorum sensing

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molecules in the bacterial system either by physical separation or chemical transformation into

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non-active molecules using clays demonstrate possible way of disrupting quorum sensing in

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bacteria which could have a possible impact on controlling bacterial ability to produce toxins or

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virulence as such functions in bacteria are control through quorum sensing.

104 105

2. Materials

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2.1 Clays and chemicals

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The clay samples were obtained from Amlan international/Oil-Dri corporation of America [13].

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Calibrin-A and Z are type of processed montmorillonite clays and their details have been


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described earlier [11, 12]. All the other chemicals were obtained from Sigma-Aldrich, USA and

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used as-received.

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3. Methods

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3.1 Clay Characterization

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Clays were characterized by X-ray diffraction (XRD) on a Bruker D2 Phaser instrument using

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Cukα as the X-ray source. A wet slide method of sample preparation was used for the analysis

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(Data not shown). A Quantachrome NOVA 4200 instrument was used to measure the total pore

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volume of Calibrin A and Calibrin Z samples by N2 adsorption/desorption method. The sample

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was degassed at 110 oC for 12 h under vacuum followed by nitrogen adsorption/desorption study

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at 77 K. Pore volume was estimated by Density Functional Theory (DFT) method of analysis

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using NovaWin 11.03. Specific surface areas of the materials were evaluated using Brunner

121

Emmett Taylor (BET) method from N2 data.

122 123

The acidity of the clay samples was measured on a Chemisorb 2720, Micromeritics instrument

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using ammonia as a probe molecule. Typically, 25–100 mg of the sample (100 mesh) was held

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between small lumps of inert quartz wool taken in a quartz tube. The sample tube was connected

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to the device and heated to 120oC, 12 h, to remove adsorbed water and other volatile impurities.

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After cooling to 25oC, dry ammonia gas [20 % NH3 balance He (UHP); 20 mL/min] was then

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passed over the sample for 15 minutes. The physically adsorbed ammonia was flushed out by He

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carrier gas at 25oC. The sample was heated from 25oC to 600oC at a heating rate of 5oC/min and

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the desorbed ammonia was measured using a thermal conductivity (TC) detector connected to

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the sample tube in a loop.

132 133

3.2 in vitro catalysis experiments

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N-(3-Oxooctanoyl)-DL-homoserine lactone was used as a model compound in this study for in

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vitro testing of clay performance. Typically, a fixed amount of aqueous lactone solution (200

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ppm) was taken in a small vial containing a fixed amount (1mg – 500 mg) of clay or solid. The

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formed suspension was then agitated at 100 rpm for 15 minutes, and successively centrifuged (a

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Thermo Scientific Sorvall Legend T+ unit) at 3,500 rpm for 30 minutes. The supernatant was

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analyzed using an Agilent 1260 Infinity HPLC equipped with diode array detection (DAD) and


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Agilent Zorbax RX-C8 column (150mm x 4.6mm x 5µm). The identity of the products was

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confirmed on an Agilent LC/MS coupled to a Bruker AmaZon X ESI-ion trap mass

142

spectrometer.

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3.3 Catalytic activity in biological system/disruption of quorum sensing in bacteria

146 147

Method 1

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Five µl of Vibrio harveyi (ATCC14126, a luminous strain) bacterial culture (prepared overnight)

149

was inoculated with 5 ml of Luria-Bertani broth (LB) + (LB + 2% NaCl) broth and at different

150

clay loadings (10 to 10,000 µg/mL) in the system. A non-luminescent Vibrio parahaemolyticus

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strain was included as an additional negative control. Bacterial culture was incubated at 30oC

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with agitation (200 rpm) for 10 h. During the culturing period, bacterial growth was monitored

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by both optical density measurements at 600 nm (BioPhotometer, Eppendorf) and by a viable

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bacterial cell count on agar plates. The luminescent emission was detected and quantified by a

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luminescence detector (MiniLumat, EG &G Berthold) every hour. Blank experiments were also

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conducted to check if LB and clay would interfere with the detection of bacterial luminescence.

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Luminescent bacteria were added to LB at different amounts of clay, and the mixtures were

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immediately subjected to luminescence detection, which indicated no variation in the detected

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luminescence upon the addition of clay to the bacterial medium, signifying no interference in

160

luminescence measurement.

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Method 2

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Five µl of Vibrio harveyi (ATCC14126, a luminous strain) bacterial culture prepared overnight

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was inoculated with 5 ml of LB+ broth and incubated at 30oC for 4 h. The cultured bacteria were

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separated (pellet) by centrifugation at 4000g for 10 min. The supernatant was collected and

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filtered through a syringe filter with a 0.45 µm pore size (PALL). The purpose of this step was to

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harvest quorum sensing molecules in the supernatant. Five ml of the filtrate was then mixed with


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different amounts of Calibrin Z to reach a final concentration of 0.5, 1, 10 and 50 mg/ml of

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Calibrin Z in the system. These suspensions were then incubated at 30oC for 1 h with agitation

169

(200 rpm). A blank filtrate containing no added clay was also included as a control in each

170

experiment. After incubation, the clays were separated from the filtrate by centrifugation at 5000

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RPM, followed by filtration of the supernatant through a 0.45 µm syringe filter as described

172

above. It is expected that different amounts of clay would impact the separation of quorum

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sensing molecules differently, thereby, delaying the bioluminescence from a culture containing

174

the least number of quorum sensing molecules. A 4 µl aliquot of a new and growing Vibrio

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harveyi bacterial culture (not yet luminescing) was inoculated into 4 ml of above filtrates, and

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the mixture was incubated at 30oC. The luminescent emission and the bacterial growth were

177

quantified by a luminescence detector (MiniLumat, EG &G Berthold) and the optical density at

178

600 nm (BioPhotometer, Eppendorf), respectively, at various time of the inoculation intervals.

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4. Results and discussion

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The removal of N-(3-Oxooctanoyl)-DL-homoserine lactone was studied in aqueous

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solutions at different temperatures (5-25oC) and at varying concentrations of lactone and clay

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amount (changing the ratio of lactone and clay), Table 1 shows one such representative data

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collected in triplicates at clay/lactone ratio of 50 (mg/mg) at 25 oC and 30 min reaction time. The

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concentration of the lactone in the reaction system was monitored using HPLC and LC-MS

186

instrumental techniques. The percentage removal (% R) term in Table 1 indicates the extent of

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lactone removal from the aqueous solution. It was observed that clay catalysts degraded the

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lactone molecule to smaller fragments on the surface acid sites on the catalyst. The structure and

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mineralogy of Calibrin clays was described previously [11-12]. Other clays used here were

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commercial samples and their structures and mineralogy were confirmed by standard

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characterization methods. In the case of precipitated silica and kaolinite, the small amount of

192

lactone removal happened totally through the adsorption process only as these materials did not

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have the required active sites/surface area for the degradation of lactone, the % lactone removal

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was 100% due to adsorption. Seemingly, the Lewis and Bronsted acid sites on the catalyst

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surface were active in degrading the lactone ring in the N-(3-Oxooctanoyl)-DL-homoserine

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lactone. As an example, Table 2 shows the structures and molecular weights of the lactone


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degradation products on Calibrin Z. Although the data shown in Table 1 was collected at 25oC,

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Calibrin clays were substantially active even at low temperature (up to 5 oC). Furthermore, there

199

existed an optimum level (amount of the solid material) to function as a catalyst, and below that

200

level the material just operated as an adsorbent. The two Calibrin products started to function as

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catalysts at a catalyst/lactone ratio of 15 (mg/mg) and above. In fact, Calibrin Z showed near

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100% catalytic degradation of lactone at a catalyst/lactone ratio of ~500.

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From the percentage lactone removal shown in Table 1 the strong activity of Calibrin

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clays for degrading the quorum sensing lactone is very clear. Generally, the surface area,

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porosity and the surface acid sites of the clay catalyst have a stronger influence on its

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performance in a catalytic reaction. The basic catalyst characterization data shown in Table 1

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correlated very well with their performance in the lactone removal and degradation under the

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given experimental conditions. The catalytic cleavage of N-(3-Oxooctanoyl)-DL-homoserine

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lactone molecule is mostly promoted by the acid sites, the number of such sites and their

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distribution in the solid material are crucial parameters to decide the progress of the reaction.

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Both the Calibrin clays have higher acidity, pore volume and surface area leading to their higher

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performance as catalysts/adsorbents in lactone removal. Silica, illite and kaolinite materials fared

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poorly in lactone removal which was probably due to their lower surface area, pore volume and

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weaker acidity. The N-(3-Oxooctanoyl)-DL-homoserine lactone used here is an organic

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molecule with an amide group linked to a saturated C8 fatty acid containing an oxo group at C3

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position (entry 6, Table 2). The molecule underwent catalytic cleavages at several positions in

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the molecule during the degradation as evidenced from the nature of the products (Table 2)

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formed and identified using LC-MS procedure described by Nievas et al. [14]. Some

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polymerized products of the original AHL molecules were also observed in LC-MS of the

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reaction mixture.

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At the next stage, we evaluated the performance of Calibrin Z clay in disrupting

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quorum sensing in a live/growing Vibrio harveyi bacteria. It is reported that the marine

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bacterium Vibrio harveyi ATCC BAA-1116, for example, channels the information of three AI

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signals into one QS cascade. Three receptors perceive these AIs, the hybrid histidine kinases

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LuxN, Lux(P)Q and CqsS, to transduce the information to the histidine phosphotransfer (HPt)

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protein LuxU via phosphorelay, and finally to the response regulator LuxO [15]. Recently, many


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other bacteria have been shown to control cell density-dependent functions through the excretion

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of and response to acyl-homoserine lactone autoinducers and the quorum sensing activity of such

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bacteria can be directly evaluated by measuring bioluminescence in bacterial cultures [16, 17].

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Besides, Vibrio harveyi is also a common pathogen causing vibriosis—a major disease of

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shrimps, fish, and shellfish that results in serious productivity and economic losses for

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aquaculture industry [18, 19].

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We used two different methods (1 and 2) to directly monitor [20, 21] the amount of

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luminescence emitted by the bacteria as the measure of catalytic quorum quenching performance

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of Calibrin Z. As shown in Figure 1, in method 1, Calibrin Z, at and above the inclusion level of

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1 mg/mL in the bacterial broth, starts to reduce the overall luminescence produced by the

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bacteria in comparison to the control (system without any added clay). In fact, at the clay

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concentrations of 10 mg/ml, the bacterial luminescence was reduced by 55% (from the area

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under the curves), respectively, which indicated a significant catalytic quorum quenching or

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interference in the overall quorum sensing of Vibrio harveyi by Calibrin Z clay. There already

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exist reports linking the bacterial population and luminescence reduction because of quorum

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quenching using different approaches [20, 21]. Importantly, as seen from Figure 2, the bacterial

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count during the measurement period was not reduced upon clay addition, confirming

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accomplishment of catalytic quorum quenching by transformation/separation of quorum sensing

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molecules or interference of quorum sensing process, and not by killing (antibacterial) of the

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bacteria in the system. Of course, the quorum sensing in bacteria is regulated by several factors

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and the presence of N-acylhomoserine lactones play a major role in this bacterial

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communication; clays could also have impacted chemical transformation and disruption of the

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overall quorum sensing process in some way or the other. The elucidation of the exact role of

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clays in impacting bioluminescence produced because of quorum sensing in Vibrio harveyi needs

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to be further investigated to confirm clay’s role in chemical transformations but direct

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interference of clays in impacting quorum sensing activity of the bacteria is confirmed from this

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study as luminescence is reduced without the loss of bacterial population in the Vibrio harveyi

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system.

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In method 2, the just formed quorum sensing molecules or their precursors in the Vibrio harveyi

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bacterial culture were first separated out in the form of a supernatant by centrifugation. The

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collected supernatant containing quorum sensing molecules was treated with different amounts


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of Calibrin Z clay and the luminescence was monitored with time. As seen in Figure 3, those

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systems that originated from clay-treated filtrates showed reduction in luminescence after 1h of

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growth, after which the intensity began to catch up. By 3 h of culture period, the luminescence

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recovered to the original level of the medium control. The amount of the clay impacted

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significantly the luminescence produced by the bacteria. Higher the clay amount, lower was the

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relative luminescence after 1 and 2 hours of bacterial cultures. The results proved that Calibrin Z

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clay caused a delay in the emission of luminescence by selectively interfering with the quorum

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sensing system of Vibrio harveyi without causing any change in bacterial population (Figure 4).

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As mentioned above, the exact role of the clay in disintegrating or transforming the quorum

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sensing molecules needs to be further explored and currently is beyond the scope of this paper.

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To the best of our knowledge, the disruption of quorum sensing molecules by catalysis of

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quorum sensing molecule is not reported thus far. As clays are regulatory compliant and safe for

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inclusion in animal feed, our approach of using clays as antagonists or catalysts to quench

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quorum sensing and thus bacterial communication could be a very promising method for

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controlling bacterial diseases caused to toxin secretion or other bacterial secretion resulting from

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quorum sensing activity.

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In conclusion, clays can be used as antagonists in quorum quenching of N-(3-

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Oxooctanoyl)-DL-homoserine lactone, a quorum sensing molecule by means of catalytic

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degradation of the lactone. Quorum sensing disruption activity of clays was also demonstrated in

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Vibrio harveyi wherein delaying of the bioluminescence gene expression was observed in these

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Gram-negative bacteria. In

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breakdown

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physicochemical properties such as structure, surface area, porosity, acidity, etc.

of

in vitro study, the performance of the clays towards catalytic

N-(3-Oxooctanoyl)-DL-homoserine

lactone

was

correlated

with

their

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Poult. Res., 2012, 21, 806-815.

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14. Nievas, F.; Bogino, P.; Sorroche F.; Giordano, W., Detection, Characterization, and

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Biological Effect of Quorum-Sensing Signaling Molecules in Peanut-Nodulating Bradyrhizobia

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Sensors 2012, 12, 2851-2873.

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15. Lorenz, N.; Shin, J.; Jung K., Activity, Abundance, and Localization of Quorum Sensing

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Receptors in Vibrio harveyi Front Microbiol. 2017; 8, 634, 1-10.

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16. Bassler, B.; Greenberg, E.; Stevens, A.; Cross-species induction of luminescence in the

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quorum-sensing bacterium Vibrio harveyi, J. Bacteriol., 1997, 179,12, 4043–4045.

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17. Fuqua, W. C., S. C. Winans, and E. P. Greenberg. Quorum sensing in bacteria: the LuxR-

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339 340 341 342 343 344


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List of Tables and Figures

356

Table 1. Physicochemical characteristics of clays and % Removal (R) of N-(3-Oxooctanoyl)-

357

DL-homoserine lactone at 25oC and 30 min reaction time (catalyst/lactone = 50).

358

Table 2. LC-MS data showing structures of various N-(3-Oxooctanoyl)-DL-homoserine lactone

359

degradation products obtained on Calibrin Z at 25oC and 30 min reaction time (clay/lactone =

360

50).

361

Figure 1. Bacterial luminescence from the culture treated at different concentrations of Calibrin

362

Z in the Vibrio harveyi culture; medium control, Vibrio parahaemolyticus. This data was

363

obtained through method 1.

364

Figure 2. Bacterial count measured as concentration (CFU/ml) of viable bacterial cells at each

365

time point from Vibrio harveyi culture at different concentrations of Calibrin Z; medium control,

366

Vibrio parahaemolyticus. This data was obtained through method 1.

367

Figure 3. Relative luminescent emission (%) Vibrio harveyi cultures treated with initial Vibrio

368

harveyi culture filtrates that were treated with various amounts of Calibrin Z. This data was

369

obtained through method 2. The relative luminesce is based of luminesce measure at zero

370

amount of clay.

371

Figure 4. Growth of Vibrio harveyi after mixing the initial Vibrio harveyi culture filtrates

372

treated with different concentrations of Calibrin Z. This data was obtained through method 2.


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373 374 375 376 377 378 379 380 381 382 Material

BETa

Pore volume,

surface area

DFTb

2

(m /g)

TPDc-NH3 acidity

%R

(150-350oC), mmol/g

(cm3/g)

Calibrin-A

141

0.32

0.025

53

Calibrin-Z

110

0.26

0.024

60

Illite

70

0.06

0.022

36

Kaolinite

32

0.04

0

5

Precipitated

340

1.0

0

25

SiO2 383

a

Brunauer–Emmett–Teller; b Density Functional Theory; c Temperature Programmed Desorption

384 385


14

Page 15 of 21


386 387 388 389

Table 1

390 391 392 393

Entry

m/z (amu)

1

86

2

102

3

114

4

141

5

157

6*

264

Proposed Structure

394 395 396 397 398 399 400 401 402 403 404 405 406


15


407 408

Page 16 of 21

*Starting (reactant) QS molecule Table 2

409 410

411 412

Figure 1

413


16

Page 17 of 21


414 415

Figure 2


17


Page 18 of 21

416 417

Figure 3

418


18

Page 19 of 21


419 420 421 422

Figure 4

423 424 425 426 427 428 429 430


19


Page 20 of 21

431

432 433 434 435

Figure : TOC Graphic


20

Page 21 of 21


436 437 438 439


21

Quorum Sensing Disruption in Vibrio harveyi Bacteria by Clay

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