Effects of micro-algae commonly used in aquaculture on acyl-homoserine lactone quorum sensing

Abstract

Quorum sensing, bacterial cell-to-cell communication with small signal molecules such as acyl-homoserine lactones, regulates the virulence of many pathogenic bacteria. Therefore, interfering with quorum sensing is currently being explored as a novel biocontrol strategy to fight bacterial infections. In this study, the effects of 19 micro-algal strains on acyl-homoserine lactone-regulated phenotypes of three reporter strains were investigated. Two freshwater micro-algae inhibited violacein production of quorum sensing reporter strain Chromobacterium violaceum CV026. Further tests using Escherichia coli JB523 showed that micro-algal extracts inhibited or stimulated quorum sensing, depending on the algal strain. One freshwater and five marine algae showed quorum sensing inhibitory activity, whereas two algae stimulated quorum sensing-regulated gene expression. Micro-algal strains that showed inhibitory activity in the previous assays also inhibited acylhomoserine lactone-regulated bioluminescence in the aquaculture pathogen Vibrio harveyi. The growth of all reporter strains was found to be unaffected by the micro-algal samples. The most promising micro-algal strain was found to be Chlorella saccharophila CCAP211/48, as its extracts inhibited quorum sensing-regulated gene expression in all three reporter strains.

1. Introduction

Bacteria and micro-algae co-exist in the aquatic ecosystem and although they are evolutionary highly distinct, studies revealed that several interactions occur between them. One type of interaction is through interfering with quorum sensing (QS) — bacterial cell-to-cell communication via the production, release and detection of small signal molecules (Fuqua et al., 2001; Jayaraman and Wood, 2008; Waters and Bassler, 2005). Different types of signal molecules are produced by bacteria, with acylated homoserine lactones (AHLs) being the most studied QS signals to date (Boyer andWisniewski-Dyé, 2009). Using this cell-to-cell communication system, bacteria regulate gene expression important for growth, survival and pathogenicity (De Kievit and Iglewski, 2000; Williams et al., 2000). The ability to interfere with QS constitutes a new biocontrol strategy since there is a link between QS and pathogenesis of many important bacterial pathogens (Donabedian, 2003; Finch et al., 1998).

QS interference has been shown to occur between algae and bacterial biofilms (reviewed by Joint et al., 2007; Rivas et al., 2010). In addition, marine macro-algae are also found to be able to disrupt QS (reviewed by Dobretsov et al., 2009 and Natrah et al., 2011). The marine macro-alga Delisea pulchra has been extensively studied for its capability to inhibit bacterial colonization through release of halogenated furanones (Dworjanyn and Steinberg, 1999; Givskov et al., 1996; Rasmussen and Givskov, 2006). Three new AHL antagonists have also been discovered from the red macro-alga Ahnfeltiopsis flabelliformis (Kim et al., 2007). Mixtures of compounds isolated from this alga inhibited AHL-regulated quorum sensing in a dose-dependent way (Liu et al., 2008). Meanwhile, minor QS-interfering activity has been detected in algae from the families Caulerpaceae, Rhodomelaceae and Galaxauraceae (Skindersoe et al., 2008). Recently, the production of QS-inhibitory metabolites has also been reported in different marine bacteria (Dobretsov et al., 2010; Kanagasabhapathy et al., 2009; Teasdale et al., 2010). In contrast to macro-algae and marine bacteria, not much is known on the effect of secondary metabolites produced by micro-algae on bacterial QS. Chlamydomonas reinhardtii is the only micro-alga that has been reported thus far to produce QS-interfering compounds (Rajamani et al., 2008; Teplitski et al., 2004).

As quorum sensing has been shown to regulate the virulence of many aquaculture pathogens (Natrah et al., 2011), quorum sensing interference by micro-algae might offer interesting opportunities for prevention of bacterial disease in aquaculture. Therefore, in this study, we have evaluated the effect of different strains of micro-algae commonly used in aquaculture on QS-regulated gene expression. The ability to interfere with AHL QS was investigated by using different bacterial reporter strains, including the aquaculture pathogen Vibrio
harveyi.

2. Materials and Methods

2.1. Reagents

N-Hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-Lhomoserine lactone (OHHL) were purchased from Sigma-Aldrich (Bornem, Belgium). All reagents were diluted using distilled water or ethanol and further diluted with algal medium. All stock solutions were kept at −20 °C. Solutions containing AHLs were buffered at pH 6.5 by the addition of 3-(N-morpholino)-propanesulfonic acid) (MOPS, Sigma).

2.2. Microalgal growth conditions

Axenic microalgal strains were obtained from the Culture Collection of Algae and Protozoa (CCAP, Dunstaffnage Marine Laboratory, Scotland), the Culture Collection of Algae University Göttingen (SAG, Germany), the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP, USA). Phaeodactylum tricornutum PT18.6 was a kind gift of Dr. W. Vyverman (Department of Plant Systems Biology, Ghent University). All strains used in this study are listed in Table 1.

Freshwater micro-algae were grown in Bold’s basal medium (BBM) and marine strains in Guillard’s F2 medium. All algae were cultured using sterile 250 ml Schott bottles, provided with 0.22 μm filtered aeration. All parameters for algal culture were kept constant (pH 7, continuous light of 100 μmol photons m−2 s −1, temperature of 24 °C and 30 g l−1 salinity for marine spp). The density of the cultures was measured using a Bürker hemacytometer and a spectrophotometer (Thermo Spectronic) at OD550 nm. Algae from three independent cultures were harvested at late exponential and late stationary phase using a centrifuge (5000 rpm for 5 min). Supernatants of the algal cultures were collected and stored at −20 °C in sterile containers. Axenity tests were done by plating the supernatant on marine agar, luria bertani agar (bacterial contamination test) and potato dextrose agar (fungal contamination test). Samples were also routinely checked microscopically at 1000× magnification with oil immersion,
immediately before harvesting.

Table 1
List of algal strains used in this study

CCAP, Culture Collection of Algae and Protozoa Dunstaffnage Marine Laboratory (Scotland, UK); SAG, Culture Collection of Algae University Göttingen (Germany); CCMP, Provasoli-Guillard National Center for Culture of Marine Phytoplankton (USA).

2.3. Preparation of extracts of algal supernatants

Extracts for QS-interfering activity were prepared according to Teplitski et al. (2004) with modifications. Ten milliliters of cell-free microalgal supernatants were mixed with an equal volume of ethyl acetate. Samples were shaken thoroughly and were centrifuged at 3000 rpm for 10 min. The ethyl acetate fractions were collected, and the extraction step was repeated twice. Samples were then rotary evaporate under reduced pressure at 30 °C water temperature and were redissolved in 100 μl of 100% acetonitrile and diluted with 300 μl distilled water. All samples were kept at −20 °C in glass sample bottles until further use.

2.4. Detection of quorum sensing-interfering activity

2.4.1. Chromobacterium violaceum CV026 violacein agar plate assay

C. violaceum CV026 is a mini-Tn5 mutant of wild type strain ATCC 31532 that is deficient in CviI (AHL synthase) and produces the purple pigment violacein in the presence of exogenous AHL (McClean et al., 1997). The reporter is most sensitive to HHL. Twenty milliliters of buffered (2 g/L MOPS) Luria-Bertani (pH 6.5) agar was mixed with HHL giving a final concentration of 10 μg/L. The solidified agar was then covered with 100 μl of C. violaceum CV026 (grown with kanamycin at 20 mg/L). 10 μl of extracts diluted four times with distilled water were spotted on the plates. Extracts of fresh algal growth medium were used as a negative control. Plates were incubated for 24 h at 27 °C and inhibition of QS-regulated violacein production was checked the next day. Stimulation of violacein production was assayed similarly, without addition of HHL into the plates.

2.4.2. Escherichia coli JB523 green fluorescent protein (GFP) microplate assay

E. coli JB523 is a highly sensitive reporter strain and contains plasmid pJBA130 that encodes stable green fluorescent protein (GFP) in the response of exogenous AHL (Andersen et al., 2001). The strain is most sensitive to OHHL. Strain JB523 was grown overnight at 28 °Cin buffered LB medium supplemented with 20 mg/L tetracycline until optical density reached approximately 1 at 550 nm. The bacteria were then diluted to OD600 of 0.1 and were mixed with OHHL standards or algal extracts (diluted six times with distilled water). Extracts of fresh algal growth medium were used as a negative control. The plate was
incubated at 28 °C for 16 h. QS-regulated GFP production was then assessed by fluorescence measurements (excitation 475 nm and emission 515 nm) using a Tecan multireader. The fluorescence was normalized for cell density of the reporter strain (OD550). Percent inhibition of QS-regulated GFP production was calculated relative to the respective medium control.

2.4.3. V. harveyi AHL-regulated bioluminescence assay

QS-regulated luminescence in V. harveyi was measured according to Henke and Bassler (2004) with slight modifications. V. harveyi JMH612, a signal receptor double mutant of strain BB120, that only produces
luminescence in response to HAI-1, an AHL (Henke and Bassler, 2004), was used as reporter. The reporter was grown overnight in marine broth (Difco) at 28 °C with shaking (100 rpm) until OD550=1. The culture was subsequently diluted with fresh marine broth (1:1000) and regrown for another six hours until luminescence reached a maximum. The reporter was then mixed with 10-fold diluted algal extract (1:1) and inoculated in triplicate in black microtiter plates. Luminescence was read every 30 min for 2 h. Extracts of fresh algal medium were used as negative control. Growth was measured by transferring each sample to a transparent microtiter plate where it was read at 550 nm using a Tecan multireader. The luminescence was normalized for cell density of the reporter strain and percent inhibition of QS-regulated luminescence was calculated relative to the respective medium control. We also tested the effect of the extracts on AI-2 and CAI-1 regulated bioluminescence by using the receptor double mutants JMH597 and JAF375, respectively (Defoirdt et al., 2008).

2.5. Statistical analysis

The collected data were from three independent algal sets (n= 3). Significant differences between medium control and individual samples were analyzed with independent sample Student’s t-test. Data that were not normally distributed were transformed to satisfy the condition of homogeneity of variance. The data that did not satisfy the parametric test were analyzed with Mann–Whitney test. Statistical analyses were done using the Statistical Package for the Social Sciences (SPSS), version 13.0.

3. Results

Reporter strain C. violaceum CV026 showed clear violacein production on agar plates containing 10 μg/L of HHL. Clear inhibition of violacein production was observed on the plates on which extracts of the freshwater algae C. saccharophila CCAP211/48 and C. vulgaris CCAP211/12 were spotted (Table 2). Furthermore, the inhibition zones were opaque, indicating that the growth of the reporter strain
was unaffected. Therefore, the lack of purple pigment was not due to bacteriostatic activity of the extracts towards the reporter strain. The assay was further slightly modified to see whether the algae produced any metabolites that could stimulate QS-regulated violacein production in CV026. In this assay, each algal extract was spotted to CV026 grown on plates without HHL addition. None of the samples showed stimulation of violacein production (data not shown). The absence of the violacein production showed that the algae did not produce agonists or that the concentrations are too low to be detected in the assay.

Table 2
Inhibitiona of quorum sensing-regulated violacein production in Chromobacterium violaceum CV026 in the presence of N-hexanoylhomoserine lactone by extracts from exponential phase and stationary phase cultures of different micro-algae.

^a + denotes inhibition; − denotes no inhibition.

^b Bold’s Basal freshwater algal medium.

^c F2 marine algal medium.

Inhibition of AHL-regulated GFP production in E. coli JB523 was observed in one freshwater alga and five marine algae, whereas one freshwater and one marine alga stimulated GFP production (Table 3). A significant decrease in QS-regulated GFP production was observed in extracts of C. saccharophila CCAP211/48 from late exponential phase. Meanwhile, another freshwater alga, C. reinhardtii CCAP11/45
stimulated QS-regulated GFP production in late exponential growth phase. No differences in GFP activity were observed for both species in extracts taken at late stationary phase. Five marine algae, Nannochloropsis CCAP849/9, Isochrysis sp. CCAP927/14, Tetraselmis suecica CCAP66/4, T. striata SAG41.85 and T. tetrathele SAG161-2C also significantly inhibited QS-regulated GFP production (Table 3).
Importantly, none of the algal extracts inhibited growth of the reporter strains. In fact, all Tetraselmis extracts were found to stimulate the growth of reporter strain JB523 (data not shown).

In order to investigate interference with the Vibrio harevyi AHL (Harveyi Autoinducer 1; HAI-1), we used the AI-2 and CAI-1 receptor double mutant JMH612, in which bioluminescence is regulated by AHL quorum sensing. The micro-algae that showed inhibitory activity in one of the previous reporters were selected for this assay. Chlorella saccharophilaCCAP211/48 consistentlyinhibited (pb0.05) AHL-regulated luminescence inV. harveyi during two hours of incubation (Table 4). Some of the other microalgal strains also showed inhibitory activities. However, in these cases, the inhibition was either insignificant or limited to the beginning of the incubation. None of the algae affected luminescence in the receptor double mutants JMH597 and JAF375, in which bioluminescence is regulated by the signal molecules AI-2 and CAI-1, respectively. This showed that inhibition of bioluminescence in AHL reporter strain JMH612 was specifically caused by interference with AHL signaling and not by interference with the bioluminescence biochemistry.

Table 3
Quorum sensing-regulated GFP production by Escherichia coli JB523 in the presence of 1 μg/L N-(3-oxohexanoyl)-L-homoserine lactone, with and without microalgal extracts collected at exponential or stationary phase. GFP production was determined by measuring specific fluorescencea. Extracts of fresh algal medium were used as controls; the specific fluorescence observed for medium extracts was set at 100% and the other values were normalized accordingly

a GFP fluorescence corrected for cell density of the reporter (Fluorescence/OD550nm).
Results are expressed as mean± standard deviation obtained for extracts of three
independent algal cultures.
b Bold’s Basal freshwater algal medium. c F2 marine algal medium. fluo, fluorescence; abs, absorbance.
⁎ Significantly different from medium control at pb0.05.
⁎⁎ Significantly different from medium control at pb0.01.

Table 4
Quorum sensing-regulated bioluminescence of Vibrio harveyi JMH612 (HAI-1 sensor+, AI-2 sensor-, CAI-1 sensor-), with and without algal extracts. Extracts of fresh algal medium were used as controls; the bioluminescence level observed for medium extracts was set at 100% and the other values were normalized accordingly.

4. Discussion

In this study, we performed the first large screen of quorum sensing interference by micro-algae, focussing on species that are commonly used in aquaculture. The ability to interfere with AHL QS was investigated by using different bacterial reporter strains, including the aquaculture pathogen V. harveyi. A preliminary screening revealed that 2 out of the 19 algae, C. saccharophila CCAP211/48 and C. vulgaris
CCAP211/12, inhibited AHL-regulated pigment production in reporter strain C. violaceum CV026. A second reporter strain, E. coli JB523, was used to measure the QS interfering effects in a more quantitative
manner. A significant decrease in QS-regulated GFP production was observed in extracts of the marine algae Nannochloropsis CCAP849/9, Isochrysis sp. CCAP927/14, T. suecica CCAP66/4, T. striata SAG41.85 and T. tetrathele SAG161-2 C and the freshwater alga C. saccharophila CCAP211/48. Meanwhile, another freshwater alga, C. reinhardtii CCAP11/45 stimulated QS-regulated GFP production. In contrast to our result, Teplitski et al. (2004) found that different fractions of C. reinhardtii strain 2137 culture filtrate taken at exponential phase stimulated LasR- and CepR-based reporters but not a LuxR-based one. This suggests that QS-interfering activity might be strain-specific rather than species-specific and that different C. reinhardtii strains might produce different QS-interfering metabolites. Production of secondary metabolites by micro-algae has been reported before to be strain- rather than species-dependent. Production of saxitoxin by the ‘red tide’ microalga Alexandrium spp., for instance, is known to be limited to certain strains (Shimizu, 1996). Unfortunately, we could not investigate the effect of C.reinhardtii strain 2137 in our experimental set-up because it grew very poorly in the medium used in our study.

The micro-algae that showed inhibitory activity in one of the previous reporters were investigated for inhibition of AHL-regulated bioluminescence in the aquaculture pathogen V. harveyi. C. saccharophila CCAP211/48 consistently inhibited AHL-regulated luminescence in V. harveyi during two hours of incubation. Some of the other microalgae also showed inhibitory activities. However, for these strains, the
inhibition was either insignificant or limited to the beginning of the incubation. The stability of the inhibitory activity observed for C.saccharophila CCAP211/48 in the V. harveyi assay indicates that this alga probably produces one or more metabolites with antagonistic activity. QS antagonistic compounds have been reported before in macro-algae, such as D. pulchra and A. flabelliformis. The compounds produced by these macro-algae were identified to be halogenated furanones (Manefield et al., 2002) and a mixture of betonicine, floridoside and isethionic acid (Liu et al., 2008). In contrast, no QS antagonistic
metabolites have been reported before in micro-algae.

The decrease of QS inhibitory activity over time in the V. harveyi assay by other micro-algae (such as Nannochloris atomus SAG14.87 and Nannochloropsis oculata CCMP525) might suggest that the QS inhibition in these strains was caused by the production of reactive compounds. Indeed, the reaction between signal molecules and reactive compounds will inactivate both the signals and the reactive
compounds. Consequently, in such a case, the level of the reactive compounds is decreasing over time, whereas the V. harveyi reporter strain is continuously producing signal molecules during the assay
(Henke and Bassler, 2004). This is not the case with reporter strain JB523 (Andersen et al., 2001), which could explain the difference between the JB523 assay and the V. harveyi assay. Macro-algae have
been shown before to produce unstable reactive compounds that interfere with QS. For example, Laminaria digitata produces oxidized halogen compounds that can deactivate AHL molecules (Borchardt et
al., 2001).

In conclusion, we found that differentmicro-algae interfere with AHL quorum sensing in Gram-negative bacteria. Because quorum sensing regulates the virulence of different aquaculture pathogens, micro-algae
that are able to interefere with quorum sensing might be interesting biocontrol agents for use in aquaculture. The most interesting strain in our study appears to be C. saccharophila CCAP211/48, as it inhibited
quorum sensing-regulated gene expression in all three reporter strains (using both unsubstituted, oxo-substituted and hydroxyl-substituted AHLs), including the aquaculture pathogen V. harveyi.

By F.M.I. Natrah, Mireille Mardel Kenmegne, Wiyoto Wiyoto, Patrick Sorgeloos, Peter Bossier, Tom Defoirdt

Reference: https://www.wellesu.com/10.1016/j.aquaculture.2011.04.038

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