Quorum Sensing Disruption in Vibrio harveyi Bacteria by Clay Materials

Article abstract of DOI:10.1021/acs.jafc.7b03918

This work describes the use of clay minerals as catalysts for the degradation of quorum sensing molecule N-(3-oxooctanoyl)-dl-homoserine lactone. Certain clay minerals as a result of their surface properties and porosity can catalytically degrade the quorum sensing molecule into smaller fragments. The disruption of quorum sensing by clay in a growing Gram-negative Vibrio harveyi bacteria culture was also studied by monitoring luminescence and population density of the bacteria, wherein quenching of bacterial quorum sensing activity was observed by means of luminescence reduction. The results of this study show that food-grade clays can be used as biocatalysts in disrupting bacterial activity in various media.

Full text of DOI:10.1021/acs.jafc.7b03918

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Letter
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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Quorum Sensing Disruption in Vibrio harveyi
Bacteria by Clay Materials
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Sajo P. Naik, * Jonathon Scholin, San Ching, Fang Chi, and Marc
Herpfer
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The Oil-Dri Innovation Center
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
20 sajo.naik@oildri.com
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Phone: +1- 847-537-7543
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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
molecule, N-(3-Oxooctanoyl)-DL-homoserine lactone. Certain clay minerals due to their surface
properties and porosity can catalytically degrade the quorum sensing molecule into smaller
fragments. The disruption of quorum sensing by clay in a growing Gram-negative Vibrio harveyi
bacteria culture was also studied by monitoring luminescence and population density of the
bacteria, wherein quenching of bacterial quorum sensing activity was observed by means of
luminescence reduction. The results of this study show that food grade clays can be used as
biocatalysts in disrupting bacterial activity in various media.
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KEYWORDS
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
bacterial activity in response to various stimuli. Quorum sensing was first reported in the study of
a marine bacteria Vibrio fischeri which exhibited bioluminescence upon bacteria reaching a high
population density [1]. Typically, bacteria biosynthesize distinctive chemicals including N-
acylhomoserine lactones (AHLs) as quorum sensing signals; specifically, AHLs, such as N-(3-
Oxooctanoyl)-DL-homoserine lactone are used by some Gram-negative bacteria in quorum
sensing signaling. The biochemical production and effectiveness of AHLs depend primarily on
the activity of Lux and LuxR protein families, respectively, which together with the quorum
sensing molecules, constitute the overall quorum sensing signaling system. After AHLs are
produced by Lux enzymes, they diffuse across bacterial membranes and accumulate in the
surroundings until critical local concentrations are reached [2]. At a given threshold
concentration, the AHL binds to LuxR response regulator, forming a complex that activates gene
expression. Some bacterial features controlled through quorum sensing include bacteria
population, antibiotic generation, bioluminescence, nitrogen-fixing gene regulation, Ti plasmid
conjugal transfer, virulent gene expression, pigment generation, bacterial swarming, and biofilm
formation [3].
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Quorum quenching is a process of disrupting the bacterial quorum system. Modulation of Lux,
LuxR and deactivation of quorum quenching molecules are some ways of accomplishing quorum
quenching. There exist several reports on the use of naturally occurring or artificially synthesized
antagonists as possible quorum quenchers [4,7]. Ideally, quorum sensing quenchers, inhibitors,
or regulators should not interfere with the normal physiological functions of bacterial cells or not
prompt bacteria to develop resistance. Although several different methods of quorum quenching
are reported, to the best of our knowledge there does not exist any biocatalytic ways involving
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
amounts of iron, magnesium, alkali metals, alkaline earths and other cations. Clay minerals exist
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
as solid acid catalysts. Acid-treated clays were used as catalysts in petroleum refining in the early
1930s [8-9]. However, their applicability was limited due to their lack of thermal stability under
high temperature fluidized catalytic cracking (FCC) conditions. Consequently, clays were
replaced by more stable synthetic catalysts, e.g. silica-alumina, zeolites, etc. in the petrochemical
industry. Besides catalysis clay minerals have several other applications as adsorbents, feed
additives, fillers, carriers, etc. [8-13]. In several animal health products, clay minerals are used as
feed additives as they are non-toxic, have high porosity and presence of active sites that can bind
a range of fungal and microbial toxins to improve the health of the animal [10-13]. In the animal
feed and health industry versatility of clays to perform more than one physiological functions is
desired.
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Herein, through in vitro studies we demonstrate that clays can adsorb and catalyze
bacterial quorum sensing molecules to break them into smaller fragments in an aqueous system.
We used N-(3 Oxooctanoyl)-DL-homoserine lactone, an AHL type quorum sensing molecule as
a model quorum sensing molecule for this in vitro study. We followed up the in vitro study by
directly monitoring the quorum quenching in Vibrio harveyi bacteria in the presence of clays
under simulated bacterial growth conditions. Bioluminescence and bacteria population were the
parameters employed to gauge the quorum sensing activity of the system. Clay minerals
functioned as adsorption/catalytic antagonists in that the AHLs responsible for quorum sensing
activity in some bacteria were either separated out by adsorption or were catalytically broken
down by the active site in clay surface. Reducing the concentration of quorum sensing
molecules in the bacterial system either by physical separation or chemical transformation into
non-active molecules using clays demonstrate possible way of disrupting quorum sensing in
bacteria which could have a possible impact on controlling bacterial ability to produce toxins or
virulence as such functions in bacteria are control through quorum sensing.
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2. Materials
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2.1 Clays and chemicals
The clay samples were obtained from Amlan international/Oil-Dri corporation of America [13].
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
used as-received.
3. Methods
3.1 Clay Characterization
Clays were characterized by X-ray diffraction (XRD) on a Bruker D2 Phaser instrument using
Cukα as the X-ray source. A wet slide method of sample preparation was used for the analysis
(Data not shown). A Quantachrome NOVA 4200 instrument was used to measure the total pore
volume of Calibrin A and Calibrin Z samples by N2 adsorption/desorption method. The sample
was degassed at 110 oC for 12 h under vacuum followed by nitrogen adsorption/desorption study
at 77 K. Pore volume was estimated by Density Functional Theory (DFT) method of analysis
using NovaWin 11.03. Specific surface areas of the materials were evaluated using Brunner
Emmett Taylor (BET) method from N2 data.
The acidity of the clay samples was measured on a Chemisorb 2720, Micromeritics instrument
using ammonia as a probe molecule. Typically, 25–100 mg of the sample (100 mesh) was held
between small lumps of inert quartz wool taken in a quartz tube. The sample tube was connected
to the device and heated to 120oC, 12 h, to remove adsorbed water and other volatile impurities.
After cooling to 25oC, dry ammonia gas [20 % NH3 balance He (UHP); 20 mL/min] was then
passed over the sample for 15 minutes. The physically adsorbed ammonia was flushed out by He
carrier gas at 25oC. The sample was heated from 25oC to 600oC at a heating rate of 5oC/min and
the desorbed ammonia was measured using a thermal conductivity (TC) detector connected to
the sample tube in a loop.
3.2 in vitro catalysis experiments
N-(3-Oxooctanoyl)-DL-homoserine lactone was used as a model compound in this study for in
vitro testing of clay performance. Typically, a fixed amount of aqueous lactone solution (200
ppm) was taken in a small vial containing a fixed amount (1mg – 500 mg) of clay or solid. The
formed suspension was then agitated at 100 rpm for 15 minutes, and successively centrifuged (a
Thermo Scientific Sorvall Legend T+ unit) at 3,500 rpm for 30 minutes. The supernatant was
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
confirmed on an Agilent LC/MS coupled to a Bruker AmaZon X ESI-ion trap mass
spectrometer.
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3.3 Catalytic activity in biological system/disruption of quorum sensing in bacteria
Method 1
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Five µl of Vibrio harveyi (ATCC14126, a luminous strain) bacterial culture (prepared overnight)
was inoculated with 5 ml of Luria-Bertani broth (LB) + (LB + 2% NaCl) broth and at different
clay loadings (10 to 10,000 ꢀg/mL) in the system. A non-luminescent Vibrio parahaemolyticus
strain was included as an additional negative control. Bacterial culture was incubated at 30oC
with agitation (200 rpm) for 10 h. During the culturing period, bacterial growth was monitored
by both optical density measurements at 600 nm (BioPhotometer, Eppendorf) and by a viable
bacterial cell count on agar plates. The luminescent emission was detected and quantified by a
luminescence detector (MiniLumat, EG &G Berthold) every hour. Blank experiments were also
conducted to check if LB and clay would interfere with the detection of bacterial luminescence.
Luminescent bacteria were added to LB at different amounts of clay, and the mixtures were
immediately subjected to luminescence detection, which indicated no variation in the detected
luminescence upon the addition of clay to the bacterial medium, signifying no interference in
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
was inoculated with 5 ml of LB+ broth and incubated at 30oC for 4 h. The cultured bacteria were
separated (pellet) by centrifugation at 4000g for 10 min. The supernatant was collected and
filtered through a syringe filter with a 0.45 µm pore size (PALL). The purpose of this step was to
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
Calibrin Z in the system. These suspensions were then incubated at 30oC for 1 h with agitation
(200 rpm). A blank filtrate containing no added clay was also included as a control in each
experiment. After incubation, the clays were separated from the filtrate by centrifugation at 5000
RPM, followed by filtration of the supernatant through a 0.45 µm syringe filter as described
above. It is expected that different amounts of clay would impact the separation of quorum
sensing molecules differently, thereby, delaying the bioluminescence from a culture containing
the least number of quorum sensing molecules. A 4 µl aliquot of a new and growing Vibrio
harveyi bacterial culture (not yet luminescing) was inoculated into 4 ml of above filtrates, and
the mixture was incubated at 30oC. The luminescent emission and the bacterial growth were
quantified by a luminescence detector (MiniLumat, EG &G Berthold) and the optical density at
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
solutions at different temperatures (5-25oC) and at varying concentrations of lactone and clay
amount (changing the ratio of lactone and clay), Table 1 shows one such representative data
collected in triplicates at clay/lactone ratio of 50 (mg/mg) at 25 oC and 30 min reaction time. The
concentration of the lactone in the reaction system was monitored using HPLC and LC-MS
instrumental techniques. The percentage removal (% R) term in Table 1 indicates the extent of
lactone removal from the aqueous solution. It was observed that clay catalysts degraded the
lactone molecule to smaller fragments on the surface acid sites on the catalyst. The structure and
mineralogy of Calibrin clays was described previously [11-12]. Other clays used here were
commercial samples and their structures and mineralogy were confirmed by standard
characterization methods. In the case of precipitated silica and kaolinite, the small amount of
lactone removal happened totally through the adsorption process only as these materials did not
have the required active sites/surface area for the degradation of lactone, the % lactone removal
was 100% due to adsorption. Seemingly, the Lewis and Bronsted acid sites on the catalyst
surface were active in degrading the lactone ring in the N-(3-Oxooctanoyl)-DL-homoserine
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,
Calibrin clays were substantially active even at low temperature (up to 5 oC). Furthermore, there
existed an optimum level (amount of the solid material) to function as a catalyst, and below that
level the material just operated as an adsorbent. The two Calibrin products started to function as
catalysts at a catalyst/lactone ratio of 15 (mg/mg) and above. In fact, Calibrin Z showed near
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
clays for degrading the quorum sensing lactone is very clear. Generally, the surface area,
porosity and the surface acid sites of the clay catalyst have a stronger influence on its
performance in a catalytic reaction. The basic catalyst characterization data shown in Table 1
correlated very well with their performance in the lactone removal and degradation under the
given experimental conditions. The catalytic cleavage of N-(3-Oxooctanoyl)-DL-homoserine
lactone molecule is mostly promoted by the acid sites, the number of such sites and their
distribution in the solid material are crucial parameters to decide the progress of the reaction.
Both the Calibrin clays have higher acidity, pore volume and surface area leading to their higher
performance as catalysts/adsorbents in lactone removal. Silica, illite and kaolinite materials fared
poorly in lactone removal which was probably due to their lower surface area, pore volume and
weaker acidity. The N-(3-Oxooctanoyl)-DL-homoserine lactone used here is an organic
molecule with an amide group linked to a saturated C8 fatty acid containing an oxo group at C3
position (entry 6, Table 2). The molecule underwent catalytic cleavages at several positions in
the molecule during the degradation as evidenced from the nature of the products (Table 2)
formed and identified using LC-MS procedure described by Nievas et al. [14]. Some
polymerized products of the original AHL molecules were also observed in LC-MS of the
reaction mixture.
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At the next stage, we evaluated the performance of Calibrin Z clay in disrupting
quorum sensing in a live/growing Vibrio harveyi bacteria. It is reported that the marine
bacterium Vibrio harveyi ATCC BAA-1116, for example, channels the information of three AI
signals into one QS cascade. Three receptors perceive these AIs, the hybrid histidine kinases
LuxN, Lux(P)Q and CqsS, to transduce the information to the histidine phosphotransfer (HPt)
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
of and response to acyl-homoserine lactone autoinducers and the quorum sensing activity of such
bacteria can be directly evaluated by measuring bioluminescence in bacterial cultures [16, 17].
Besides, Vibrio harveyi is also a common pathogen causing vibriosis—a major disease of
shrimps, fish, and shellfish that results in serious productivity and economic losses for
aquaculture industry [18, 19].
We used two different methods (1 and 2) to directly monitor [20, 21] the amount of
luminescence emitted by the bacteria as the measure of catalytic quorum quenching performance
of Calibrin Z. As shown in Figure 1, in method 1, Calibrin Z, at and above the inclusion level of
1 mg/mL in the bacterial broth, starts to reduce the overall luminescence produced by the
bacteria in comparison to the control (system without any added clay). In fact, at the clay
concentrations of 10 mg/ml, the bacterial luminescence was reduced by 55% (from the area
under the curves), respectively, which indicated a significant catalytic quorum quenching or
interference in the overall quorum sensing of Vibrio harveyi by Calibrin Z clay. There already
exist reports linking the bacterial population and luminescence reduction because of quorum
quenching using different approaches [20, 21]. Importantly, as seen from Figure 2, the bacterial
count during the measurement period was not reduced upon clay addition, confirming
accomplishment of catalytic quorum quenching by transformation/separation of quorum sensing
molecules or interference of quorum sensing process, and not by killing (antibacterial) of the
bacteria in the system. Of course, the quorum sensing in bacteria is regulated by several factors
and the presence of N-acylhomoserine lactones play a major role in this bacterial
communication; clays could also have impacted chemical transformation and disruption of the
overall quorum sensing process in some way or the other. The elucidation of the exact role of
clays in impacting bioluminescence produced because of quorum sensing in Vibrio harveyi needs
to be further investigated to confirm clay’s role in chemical transformations but direct
interference of clays in impacting quorum sensing activity of the bacteria is confirmed from this
study as luminescence is reduced without the loss of bacterial population in the Vibrio harveyi
system.
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In method 2, the just formed quorum sensing molecules or their precursors in the Vibrio harveyi
bacterial culture were first separated out in the form of a supernatant by centrifugation. The
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
systems that originated from clay-treated filtrates showed reduction in luminescence after 1h of
growth, after which the intensity began to catch up. By 3 h of culture period, the luminescence
recovered to the original level of the medium control. The amount of the clay impacted
significantly the luminescence produced by the bacteria. Higher the clay amount, lower was the
relative luminescence after 1 and 2 hours of bacterial cultures. The results proved that Calibrin Z
clay caused a delay in the emission of luminescence by selectively interfering with the quorum
sensing system of Vibrio harveyi without causing any change in bacterial population (Figure 4).
As mentioned above, the exact role of the clay in disintegrating or transforming the quorum
sensing molecules needs to be further explored and currently is beyond the scope of this paper.
To the best of our knowledge, the disruption of quorum sensing molecules by catalysis of
quorum sensing molecule is not reported thus far. As clays are regulatory compliant and safe for
inclusion in animal feed, our approach of using clays as antagonists or catalysts to quench
quorum sensing and thus bacterial communication could be a very promising method for
controlling bacterial diseases caused to toxin secretion or other bacterial secretion resulting from
quorum sensing activity.
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In conclusion, clays can be used as antagonists in quorum quenching of N-(3-
Oxooctanoyl)-DL-homoserine lactone, a quorum sensing molecule by means of catalytic
degradation of the lactone. Quorum sensing disruption activity of clays was also demonstrated in
Vibrio harveyi wherein delaying of the bioluminescence gene expression was observed in these
Gram-negative bacteria. In in vitro study, the performance of the clays towards catalytic
breakdown of N-(3-Oxooctanoyl)-DL-homoserine lactone was correlated with their
physicochemical properties such as structure, surface area, porosity, acidity, etc.
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List of Tables and Figures
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Table 1. Physicochemical characteristics of clays and % Removal (R) of N-(3-Oxooctanoyl)-
DL-homoserine lactone at 25oC and 30 min reaction time (catalyst/lactone = 50).
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Table 2. LC-MS data showing structures of various N-(3-Oxooctanoyl)-DL-homoserine lactone
degradation products obtained on Calibrin Z at 25oC and 30 min reaction time (clay/lactone =
50).
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362
363
Figure 1. Bacterial luminescence from the culture treated at different concentrations of Calibrin
Z in the Vibrio harveyi culture; medium control, Vibrio parahaemolyticus. This data was
obtained through method 1.
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365
366
Figure 2. Bacterial count measured as concentration (CFU/ml) of viable bacterial cells at each
time point from Vibrio harveyi culture at different concentrations of Calibrin Z; medium control,
Vibrio parahaemolyticus. This data was obtained through method 1.
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369
370
Figure 3. Relative luminescent emission (%) Vibrio harveyi cultures treated with initial Vibrio
harveyi culture filtrates that were treated with various amounts of Calibrin Z. This data was
obtained through method 2. The relative luminesce is based of luminesce measure at zero
amount of clay.
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Figure 4. Growth of Vibrio harveyi after mixing the initial Vibrio harveyi culture filtrates
treated with different concentrations of Calibrin Z. This data was obtained through method 2.
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Material
BETa
surface area
(m2/g)
Pore volume,
DFTb
TPDc-NH3 acidity
% R
(150-350oC), mmol/g
(cm3/g)
0.32
0.26
0.06
0.04
1.0
Calibrin-A
Calibrin-Z
Illite
141
110
70
0.025
0.024
0.022
0
53
60
36
5
Kaolinite
32
Precipitated
SiO2
340
0
25
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385
a Brunauer–Emmett–Teller; b Density Functional Theory; c Temperature Programmed Desorption
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402
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404
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406
Table 1
Entry
1
m/z (amu)
86
Proposed Structure
2
102
3
4
5
114
141
157
6*
264
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*Starting (reactant) QS molecule
Table 2
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure : TOC Graphic
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