Quorum Sensing Signals Produced by Heterotrophic Bacteria in Black Band Disease (BBD) of Corals and Their Potential Role in BBD Pathogenesis

Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease

Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome

Black band disease infection on a colony of <i>Diploria strigosa</i> on a reef of Curaçao.

<p>The dark-colored black band disease microbial mat separates apparently healthy coral tissue from white, denuded coral skeleton. Photograph provided by Abigael Brownell.</p

Reporter assay results and sequencing data for the eleven bacterial isolates that tested positive for AHL production.

1<p>“+” indicates a positive assay result and a “−” indicates a negative result.</p>2<p>Although BBD-FTL-6j and BBD-FTL-8c shared the same closest relative in GenBank, these isolates showed differing colony morphologies and were isolated from two different BBD samples on three separate coral colonies.</p>3<p>Culture fluids of isolates not tested with the <i>C. violaceum</i> CV026 reporter strain.</p>4<p>Isolates not tested with the <i>A. tumefaciens</i> NTL4(pZLR4) reporter strain.</p>5<p>Not detectable. These isolates inhibited growth of the <i>C. violaceum</i> CV026 reporter strain in the patch tests, eliciting an area of clear, cell-free agar in the assay plate surrounding the bacterial patch. Additionally, isolate HSML-FTL-10a produced a dark purple pigment.</p><p>Reporter assay results and sequencing data for the eleven bacterial isolates that tested positive for AHL production.</p

Isolates that elicited inhibition and stimulation of growth according to isolate source and production of AHLs or AI-2 activity based on reporter strain data and DPD (LC-MS/MS).

1<p>Not tested. These strains were not analyzed for DPD production using LC-MS/MS (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108541#pone-0108541-t006" target="_blank">Table 6</a>).</p>2<p>The percent value here is the percent of the total challenges (144 BBD strains, 108 BSML strains, and 90 HSML strains) that resulted in inhibition or stimulation of growth in each isolate source category.</p><p>Isolates that elicited inhibition and stimulation of growth according to isolate source and production of AHLs or AI-2 activity based on reporter strain data and DPD (LC-MS/MS).</p

Correlations between autoinducer production and inhibition of growth.

1<p>Percent of 81 assays that resulted in growth inhibition. Isolate HSML-FTL-10a not included (see text).</p><p>Correlations between autoinducer production and inhibition of growth.</p

Light production of the media control wells and media reference wells in the AI-2 reporter assay.

<p>The medium control curves indicate that the three growth media used in this study did not stimulate light production prior to self-induction by the <i>Vibrio harveyi</i> BB170 reporter strain. The luminescence of the medium reference wells, which contained sterile growth media, remained minimal over the course of the AI-2 reporter assay, although some minor increases in light were measured in these wells at the end of the assay due to light contamination from adjacent wells. Arrow indicates time of self-induction by the reporter strain, after which the luminescence of the reporter strain increases rapidly.</p

AHLs detected via LC-MS/MS after 24 hour incubation.

1<p>Results are an average of four samples (two cultures sampled twice).</p>2<p>Results are an average of six samples (three cultures injected twice).</p>3<p>Percentages represent relative abundance within the culture.</p>4<p>AHLs are comprised of a homoserine lactone ring attached to an acyl side chain (generally between 4–20 carbons in length) and may have a keto or hydroxy substituent at the C3-position. Abbreviations: C<i>X</i> = AHL contains “<i>X</i>” carbon molecules in the acyl chain; 3OC<i>X</i> – AHL has a keto substituent at the C3-position; 3OHC<i>X</i> – AHL has a hydroxyl substituent at the C3-position.</p>5<p>Error is reported at standard deviation.</p>6<p>Not detected.</p>7<p>Isolate BBD-FLK-1M2 was identified through 16S rRNA gene sequencing as a 100% match to <i>Ferrimonas</i> sp. EF3B-B688 (Accession No. KC545309.1). This isolate tested negative in the <i>Chromobacterium violaceum</i> CV026 patch test and was not tested using the <i>Agrobacterium tumefaciens</i> NTL4(pZLR4) assay.</p><p>AHLs detected via LC-MS/MS after 24 hour incubation.</p

Autoinducer-2 activity detected using the <i>Vibrio harveyi</i> BB170 reporter strain presented according to isolate type.

<p>Autoinducer-2 activity detected using the <i>Vibrio harveyi</i> BB170 reporter strain presented according to isolate type.</p