Autoinducer-2 bioassay is a qualitative, not quantitative ... - CiteSeerX

Journal of Microbiological Methods 66 (2006) 497 – 503 www.elsevier.com/locate/jmicmeth

Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose Yevgeniy Turovskiy, Michael L. Chikindas ⁎ Department of Food Science, Rutgers, the State University of New Jersey, New Brunswick, NJ 08901, USA Received 11 January 2006; received in revised form 2 February 2006; accepted 2 February 2006 Available online 27 March 2006

Abstract Autoinducer-2 (AI-2) is a cell-to-cell signaling molecule which is thought to be utilized for quorum sensing processes by a variety of prokaryotic species. This molecule is usually detected using a so-called autoinducer bioassay, which relies on the ability of a Vibrio harveyi reporter strain to produce light in response to AI-2. However, as previously reported, the presence of glucose in the sample can inhibit the bioluminescence of the reporter strain, a fact that is often ignored by investigators. Our data suggest that the presence of glucose in concentrations below that required for the inhibition of bioluminescence may lead to incorrect AI-2 readings and produce misleading (false-positive) results. Our findings also suggest that even if all the limitations of this bioassay are considered, the large standard deviation of the method allows only for a qualitative and not quantitative interpretation of the obtained results. © 2006 Elsevier B.V. All rights reserved. Keywords: Nisin; Listeria monocytogenes; Stress Response; AI-2; Quorum sensing; Autoinducer-2; ATR; Acid Tolerance Response

1. Introduction Microorganisms utilize a variety of pheromones to coordinate their activities on a population level. These molecules are called autoinducers and most of them are very species-specific (Smith et al., 2004). Autoinducer-2 is unique in that it is sometimes reported to be involved in interspecies communication (Federle and Bassler, 2003). This signaling molecule was first discovered in Vibrio harveyi, a marine organism which can float freely in the ocean or live in symbiotic association with some marine animals. V. harveyi utilizes cell-to-cell communication for the regulation of the genes responsible for ⁎ Corresponding author. Tel.: +1 732 932 9611x218; fax: +1 732 932 6776. E-mail address: [email protected] (M.L. Chikindas). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.02.001

bioluminescence. Biosynthesis of AI-2 in V. harveyi is dependent on luxS. Genomes of more than 50 bacterial species have homologues of V. harveyi's luxS, and the production of AI-2 was detected in a number of these species (Miller et al., 2004). LuxS catalyses formation of 4,5-dihydroxy-2,3pentanedione (DPD), the AI-2 precursor molecule, from S-ribosylhomocysteine (SRH). DPD is unstable, and can spontaneously rearrange and form a number of inter-convertible molecules with AI-2-like activity (Alfaro et al., 2004; Miller et al., 2004). AI-2(s) is/are commonly detected through the Autoinducer-2 bioassay which was developed more than a decade ago. The assay is based on the ability of V. harveyi BB170 to specifically bioluminate in response to AI-2. At lower cell-densities of BB170 (106 – 107 CFU/ml), the bioluminescence can be detected in

498

Y. Turovskiy, M.L. Chikindas / Journal of Microbiological Methods 66 (2006) 497–503

response to the added AI-2 (Bassler et al., 1994). This method was utilized in a number of studies in an attempt to characterize the mode of AI-2 production in various microorganisms. For example, Cloak et al. (2002) monitored AI-2 production by various foodborne pathogens in several food products at different temperatures. AI-2 production by the microorganisms was detected in milk and in chicken broth, but not in apple juice. Lu et al. (2004) used the AI-2 bioassay to detect the possible AI-2-like activity which may be associated with various foods and found that some foods can indeed induce bioluminescence in BB170, presumably because of molecules in the food which mimic AI-2 structurally. Back in the early 1970s, it was discovered that 5g/l of glucose (final concentration for 1010 CFU/ml) inhibits bioluminescence in V. harveyi (Nealson et al., 1972). It was proposed that this inhibition happens on the transcriptional level of the Vibrio's luciferase biosynthesis, through a catabolite repression mechanism. This finding is largely disregarded when V. harveyi is utilized for the detection of AI-2. In our study we further investigate the validity of the Autoinducer-2 bioassay, and also demonstrate how data can be misinterpreted if the pitfalls of this assay are overlooked. 2. Materials and methods 2.1. Bacterial strains, growth conditions and culture media Listeria monocytogenes Scott A was obtained from our culture collection. All the strains of V. harveyi were a gift from Dr. Bassler, Princeton University. V. harveyi BB120 is a wild type strain. We used it as a source of external AI-2 in our experiments (AI-2+). The V. harveyi MM30 strain is a luxS deletion mutant; it was used as a corresponding negative control (AI-2−). V. harveyi BB170 is a reporter strain, which specifically responds to AI-2 by bioluminescence. The stocks of all the cultures were kept at − 80°C in liquid media containing 15% v/v of glycerol. The working stock was prepared by streak-plating these frozen cells onto a solid agar media and by growing them. Then, cells from a single colony were inoculated into a fresh liquid media and grown overnight. Change in the turbidity of the culture was detected with MRX TC computer-controlled microplate reader (Dynex Technologies). Cell counts were taken at predetermined time points in order to confirm the progress of the microbial growth and the transition of the population from one growth phase into another.

Modified BHI (mBHI) medium used to grow L. monocytogenes was formulated specifically for the purpose of the Autoinducer-2 bioassay (Zhao and Montville, 2006). In order to standardize the conditions of our experiments, it was supplemented with the same salts which are present in Autoinducer Bioassay media, and therefore contained 37 g of BHI (Difco, MI), 12.5 g of NaCl, 12.3 g of MgSO4 and 10ml of 0.1 M L-arginine per 1 l of water. The stock solutions of all the ingredients were sterilized separately and then mixed together after being completely cooled down. The 0.1 M L-arginine stock solution was filter-sterilized through 0.2 μm membrane filters (NALGENE). When necessary, a modified BHI medium was buffered with 10mM KPi (final concentration). Autoinducer Bioassay (AB) medium composition (Bassler et al., 1994) was modified (simplified) from the original by the author (Bassler et al., 1994) and contained 17.5 g NaCl, 12.3 g MgSO4 and 2.0 g Casamino acids (Difco, MI) per 1 l of water (Bassler, personal communication). These ingredients were dissolved and the pH of the solution was brought up to 7.5 with 3 N NaOH. After being sterilized and completely cooled down, the following ingredients were added from separate sterile stocks: 10 ml of 1 M KPi (pH 7.0), 10ml of 0.1 M L-arginine and 10ml of Glycerol. Marine Agar (MA) was used for the proliferation of Vibrio strains. It was prepared by the addition of 1.5% agar w/v to the Marine Broth (Difco, MI). Trypticase Soy Agar (TSA) was used for the growth of Listeria cells. Nisin stock solution was prepared by dissolving 10mg of 2.5% commercial nisin preparation (SIGMA) in 1ml of nisin diluent (hydrochloric acid solution, pH 1.7), and by filter sterilizing it with a 0.2 μm membrane filter (NALGENE). Cell free (CF) supernatants were collected from bacterial cultures by centrifugation at 5000 g for 10 min and then filter-sterilized with 0.2 μm membrane filters (NALGENE). Autoindicer-2 bioassay was largely conducted according to Bassler et al. (1994). In brief, V. harveyi BB170 was grown for 16 h in AB media and then diluted 5000 times in fresh AB media to obtain 105 CFU/ml. One ml of the cell-free sample tested for the presence of AI-2 was added to 9ml of these cells, thoroughly mixed and then incubated at 30 °C with agitation (140 RPM). Bioluminescence measurements were taken every 30min with Luminoscan TL plus (ThermoLabsystems). Measurements taken after 5.5 h of incubation were normalized to the positive control (CF supernatant from


are no reports about the function of this gene in Listeria spp. L. monocytogenes Scott A was grown for 27 h and cell-free supernatants were collected at the various growth stages, starting from the mid-exponential phase and ending at the late stationary phase. Using the AI-2 bioassay, these cell-free supernatants were analyzed for the presence of Autoinducer-2. Under the experimental conditions, the population of Listeria cells transitioned through a brief lag phase, and after 12.5 h of logarithmic growth, it reached the stationary phase. We have chosen to monitor AI-2 presence in the culture after 9, 10, and 11h of growth (mid-exponential growth phase), after 12.5h of growth (late-exponential growth phase), and after 14 and 27 h of growth (stationary phase). Autoinducer-2 could not be detected in the culture after 9, 10 and 11h of incubation. This molecule's production suddenly emerged at its peak when the population transitioned into the stationary phase (after 12.5 h of growth). As determined with the AI-2 bioassay, the level of AI-2 in the cell-free supernatant of Scott A remained largely unchanged all throughout the stationary phase (Fig. 1). Autoinducers are reported as produced by microorganisms in a specific growth phase (Winzer et al., 2002). In our experiments, we did not observe any gradual AI-2 accumulation in the mid-exponential growth phase; the molecule could only be detected at the onset of the stationary growth phase (after 12.5 h of growth). This pattern of AI-2 release into media correlates well with the hypothesis that it indeed serves as an autoinducer in L. monocytogenes. Nonetheless, as soon as we became aware of the fact that the

the overnight culture of BB120) and then expressed per CFU/ml of V. harveyi reporter strain. Glucose bio-removal method was conducted as follows: V. harveyi MM30 was grown in AB media overnight at 30 °C with agitation (140 RPM). The culture was transferred into 15 ml centrifuge tubes (10 ml of the culture into each) and then centrifuged for 10 min at 4500g. Supernatant from each tube was decanted and cells were re-suspended in the neutralized cell-free supernatant collected from L. monocytogenes Scott A after 9, 10, 11, and 14 h of growth. As a negative control, the cells were also re-suspended in the mBHI media. Cells were then incubated at 30 °C for 16h with agitation (140 RPM). Cell-free supernatants were collected. They were neutralized with a 3N potassium hydroxide solution and were kept at −20°C for a maximum of 10 days, before being used. Each experiment was performed at least two times in triplicates and t test was applied to determine the statistical significance of the results (some experiments were conducted multiple times; see Results for details). 3. Results 3.1. Glucose can mimic AI-2-like activity or can totally inhibit it during the AI-2 bioassay Since it has been reported in the literature that AI-2like activity is associated with certain foods (Lu et al., 2004), we decided to investigate the possible role of AI2 in cell-to-cell communication of the foodborne pathogen L. monocytogenes Scott A. Comparative genome analysis of this organism indicates the presence of luxS in its genome (Glaser, 2001), but to-date there

0

2

4

6

499

8

10

12

14

16

2.0

1.5

1.0 0.8

1.0 0.6 0.5

OD630

Bioluminescence (AU) or Glucose (g/l)

1.2

0.4 0.2

0.0 9 10 11

14

Time (h)

Fig. 1. AI-2 (shown in bars) is not detected in supernatants from early growth stages (●) of Scott A unless glucose (■) is removed (see Fig. 2). Cellfree supernatants were collected from the L. monocytogenes culture after 9, 10, 11, 12.5, 14 and 27 h of growth. AI-2 was only detected after 12.5h of growth and remained relatively constant during 27h of observation (some data points are omitted).

500

Y. Turovskiy, M.L. Chikindas / Journal of Microbiological Methods 66 (2006) 497–503 0

2

6

4

10

8

12

14

16

2,0

1,5

1,0 6,0

0,5

5,5

0,0

5,0

0,8 0,6

OD630 (nm)

1,0 6,5

pH values

Bioluminescence (AU) or Glucose (g/l)

1,2 7,0

0,4 0,2 9 10 11

14

Time (h)

Fig. 2. Glucose removal from CF supernatants of L. monocytogenes reveals the presence of AI-2 in the mid-log growth phase. The cell-free samples for AI-2 detection were collected at 9, 10, 11, and 14h of L. monocytogenes incubation, and neutralized. The non-AI-2 producer V. harveyi strain MM30 was used to metabolize the remaining glucose and other medium components which could possibly influence the assay. The lower pH (▴) of the 9 and 10 h samples is due to the higher amount of nutrients available for utilization by MM30 cells in these samples. All the samples were neutralized once again prior to AI-2 assay. When glucose (■) is removed, AI-2 (shown in bars) can be detected starting from the mid-exponential growth phase (●).

glucose (Fig. 2).This process was also reflected through the change in the pH. V. harveyi is able to utilize glucose as well as other sugars (Berkeley et al., 1994), and this particular strain did not produce AI-2 because it is a luxS deletion mutant. When these ‘glucose-depleted’ CF supernatants from L. monocytogenes were again tested for the presence of AI-2, a totally different picture emerged (Fig. 2). The very same supernatants displayed AI-2-like activity in the mid-exponential growth phase after they became

determination of AI-2 presence could be masked and/or interfered with by the remaining glucose and possibly by other components of rich media, we exercised various approaches to remove these media components. To eliminate the possible interference of media components with AI-2 detection, we utilized the following technique. V. harveyi MM30 (AI-2− strain) was inoculated into the CF supernatants from L. monocytogenes. While growing in these supernatants, MM30 consumed the remaining nutrients; in particular

1.0

0.8

control+

Bioluminescence normalized to

1.2

0.6

0.4

0.2

m B H I, 70 %

0% I, H B m

m B H I, 30 %

ro nt co

co

nt

ro

l+

l-

0.0

Fig. 3. The components of fresh mBHI media hinder the bioluminescence of the V. harveyi reporter strain in response to the added AI-2. In order to elucidate the effect of the rich medium's components on the bioluminescent response of BB170, the fixed amount of the CF supernatant from BB120 (source of AI-2) was mixed with various quantities of fresh mBHI medium. Fresh AB medium was used as a diluent for mBHI. AB media has no glucose and has the same amount of salts as mBHI so osmotic pressure of the sample is kept consistent. The 30% solution of cell-free supernatant from BB120 without mBHI induced the bioluminescent response (mBHI, 0%). While, the same solution which contained 70% or 30%mBHI (mBHI, 70%) and (mBHI, 30%) respectively, did not induce any bioluminescence in response to AI-2.


501

(Nealson et al., 1972) and is still neglected by some researchers (Fig. 3). DeKeersmaecker and Vanderleyden (2003) reported that 2 g/l of glucose in the sample being tested through the AI-2 bioassay will totally inhibit the bioluminescent response of the reporter strain to AI-2. This is in agreement with our observation. However, when we further decreased the glucose concentration in the sample that contained AI-2, the effect was rather opposite. The response of BB170 to AI-2 was noticeably magnified when samples contained low

depleted of glucose. A full AI-2-like activity could be observed after 11 h of growth and lesser activity could be observed after 9 and 10 h of growth. In order to confirm the inhibitory effect of mBHI media components, fresh mBHI medium was mixed with CF supernatant from V. harveyi BB120 (AI-2+ strain) in various pre-determined proportions. Even 30% of the fresh media in the sample which contained AI-2 was able to inhibit the sample's AI-2-like activity during the Autoinducer-2 bioassay. Our results confirm the observation which was reported more than 30years ago

A 14

normalized to control+

Relative bioluminescence

16

12 10 8 6 4 2

no glucose added

-4 4e 6.

-3 2e

6 3.

01 0.

08 0.

4 0.

2

nt co

co

nt

ro

ro

l+

l-

0

final glucose concentration (g/l) after it was added to control +

B 12 10

to control+

Bioluminescence normalized

14

8 6 4 2

g

3g 0. 0

06 0.

3g .1

5g 0. 2

5g 0.

1g

2g

ro l nt co

co

nt

ro

l+

-

0

Fig. 4. Glucose overshadows bioluminescent response of the reporter strain to AI-2. (A) The effect of glucose on Autoinducer-2 bioassay. The cellfree supernatant from V. harveyi BB120 with sterile distilled water added to it (10% of the total volume) was used as a positive control for the assay (control+). The cell-free supernatant from MM30 (AI-2- strain) was used as a negative control (control−). To determine the effect of glucose on the assay, instead of water, we added a glucose solution to the positive control (10% of the volume). The samples which contained 2.0g/l–0.4 g/l of glucose exhibited much lesser AI-2-like activity, while the samples which contained 0.08g/l–0.0032g/l of glucose exhibited a much higher AI-2-like activity than control+. (B) Pure glucose solution in the concentrations 0.25g/l–0.03g/l indirectly promotes signal enhancement inducing the bioluminescence of the reporter strain in the course of the assay.

502


concentrations of glucose. For instance, the bioluminescence induced in BB170 cells was 20 times higher than the positive control when 0.08g/l glucose was present in the tested sample containing AI-2 (Fig. 4A). The results were even more striking when progressively lower concentrations of glucose were added to the CF supernatant from MM30 (AI-2−) which was then assayed for AI-2 presence. The sample containing 0.25 g/l of glucose exhibited almost 100% AI-2-like activity, and the sample containing 0.06 g/l of glucose exhibited a 10-fold higher AI-2-like activity, than the positive control (AI-2+ sample) (Fig. 4B). 3.2. AI-2 bioassay: qualitative but not a quantitative method due to the high standard deviation In an attempt to make a quantitative comparison between the AI-2-like activities of two samples of supernatants collected from L. monocytogenes, we grew the foodborne pathogen, collected cell-free supernatants and then repeatedly tested these supernatants for the presence of AI-2. The AI-2 bioassay was conducted three times, using the very same samples (Fig. 5). Although the standard deviation between replicates within a single experiment was 1–5%, the corresponding bioluminescence values generated in separate experiments differed substantially (standard deviation 30– 40%). In total, for this particular experiment the bioluminescence assay was conducted eight times, in triplicates. Our results suggest that 2 to 3-fold differences

in AI-2-like activity between samples cannot be considered as noteworthy. 4. Discussion AI-2 bioassay is commonly conducted as described in Methods (Bassler et al., 1994). In brief, V. harveyi BB170 is grown overnight then diluted and samples are added to this culture, 10% of the total volume. The bioluminescent measurement with the most pronounced difference between positive and negative controls is commonly considered the most informative/noteworthy. This time point is usually reached after 4–5 h of incubation (Bassler et al., 1994; Surette and Bassler, 1998; Cloak et al., 2002; Lu et al., 2004). During the time of incubation, cells grow and consume nutrients from the medium. Glucose can support the growth of V. harveyi to much higher levels than glycerol (Nealson and Hastings, 1972), which is the source of carbon in AB media. Our data indicate that as little as 0.125 g/l of glucose in the sample will stimulate the growth of BB170 about 7-fold in the duration of the assay. At higher population densities, V. harveyi BB170 will produce enough indigenous AI-2 to stimulate a bioluminescent ‘self’-response (Bassler et al., 1994). The bioluminescent response of BB170 should be compared exclusively at time points when the cells are at the same population density, and when the glucose concentration in the sample is below the inhibitory level. Samples being tested for AI-2 should have a neutral pH, because the acidity of the sample may interfere with the

normalized to positive control

Relative bioluminescence per CFU/ml

1.0

0.8

0.6

0.4

0.2

co

nt

ro

l-

ni s-

ni s+

0.0

Fig. 5. Autoinducer-2 bioassay conducted three times with the same samples. Listeria monocytogenes Scott A was grown for 16h in the presence of nisin (nis+) and without it (nis−). The cell-free supernatants from these two cultures were collected as described in the methods section, neutralized and transferred into Eppendorf tubes. The samples were kept at − 20°C for a maximum of 12days. Results were expressed as CFU/ml and were normalized to the positive control, the cell-free supernatant from BB120.


assay as well (DeKeersmaecker and Vanderleyden, 2003). Our results suggest that even when all these factors are taken into account, the high variability of this method makes it, at best, qualitative only. Acknowledgement This research was supported (in part) by the New Jersey Agricultural Experiment Station Project #10152 through U.S. Hatch Act funds. The authors thank Dr. Bassler for providing the strains, for providing the composition for the simplified AB medium and for the generous suggestions regarding the method. References Alfaro, J.F., Zhang, T., Wynn, D.P., Karschner, E.L., Zhou, Z.S., 2004. Synthesis of LuxS inhibitors targeting bacterial cell–cell communication. Org. Lett. 6, 3043–3046. Bassler, B.L., Wright, M., Silverman, M.R., 1994. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13, 273–286. Berkeley, R.C.W., Bock, E., Boone, D.R., Brenner, D.J., Castenholz, R.W., Colman, G., et al., 1994. Bergey's Manual of Determinative Microbiology, Ninth edition. In: Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T. (Eds.). Williams & Wilkins, Baltimore, MD, pp. 192–194. Cloak, O.M., Solow, B.T., Briggs, C.E., Chen, C.Y., Fratamico, P.M., 2002. Quorum sensing and production of autoinducer-2 in

503

Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods. Appl. Environ. Microbiol. 68, 4666–4671. DeKeersmaecker, S.C., Vanderleyden, J., 2003. Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter. Microbiology 149, 1953–1956. Federle, M.J., Bassler, B.L., 2003. Interspecies communication in bacteria. J. Clin. Invest. 112, 1291–1299. Glaser, P., 2001. Comparative genomics of Listeria species. Science 294, 849–852. Lu, L., Hume, M.E., Pillai, S.D., 2004. Autoinducer-2-like activity associated with foods and its interaction with food additives. J. Food Prot. 67, 1457–1462. Miller, S.T., Xavier, K.B., Campagna, S.R., Taga, M.E., Semmelhack, M.F., Bassler, B.L., Hughson, F.M., 2004. Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorumsensing signal AI-2. Mol. Cell 15, 677–687. Nealson, K.H., Hastings, J.W., 1972. The inhibition of bacterial luciferase by mixed function oxidase inhibitors. J. Biol. Chem. 247, 888–894. Nealson, K.H., Eberhard, A., Hastings, J.W., 1972. Catabolite repression of bacterial bioluminescence: functional implications. Proc. Natl. Acad. Sci. U. S. A. 69, 1073–1076. Smith, J.L., Fratamico, P.M., Novak, J.S., 2004. Quorum sensing: a primer for food microbiologists. J. Food Prot. 67, 1053–1070. Surette, M.G., Bassler, B.L., 1998. Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc. Natl. Acad. Sci. U. S. A. 95, 7046–7050. Winzer, K., Hardie, K.R., Williams, P., 2002. Bacterial cell-to-cell communication: sorry, can't talk now — gone to lunch! Curr. Opin. Microbiol. 5, 216–222. Zhao, L., Montville, T.J., 2006. Evidence for quorum sensing in Clostridium botulinum 56A. Lett. Appl. Microbiol. 42, 54–58.