Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer Beth L. Zimmer, Amanda L. May, Chinmayee D. Bhedi, Stephen P. Dearth, Carson W. Prevatte, Zoe Pratte, Shawn R. Campagna, Laurie L. Richardson mail Published: September 30, 2014 •DOI: 10.1371/journal.pone.0108541 Article About the Authors Metrics Comments Related Content Abstract Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract 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. Figures 1234 Citation: Zimmer BL, May AL, Bhedi CD, Dearth SP, Prevatte CW, et al. (2014) Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer. PLoS ONE 9(9): e108541. doi:10.1371/journal.pone.0108541 Editor: Fabiano Thompson, Universidade Federal do Rio de Janeiro, Brazil Received: March 6, 2014; Accepted: August 29, 2014; Published: September 30, 2014 Copyright: © 2014 Zimmer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the NSF (grant no. OCE-1208784 to LLR and SRC and OCE-1061352 to SRC) and by Florida International University. The websites are www.nsf.gov and www.fiu.edu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. First author Beth Zimmer is employed by the commercial company Atkins North America. Atkins North America provided support in the form of salary for author BZ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section. Publication of this article was funded in part by Florida International University and in part by the University of Tennessee, Knoxville, Open Access Publishing Funds. Competing interests: First author Beth Zimmer is employed by the commercial company Atkins North America. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. Introduction Coral diseases are widely believed to play a key role in the deterioration of coral reefs on a global basis , , with black band disease (BBD) identified as one of the major coral diseases contributing to this decline , . BBD is easily distinguished in the reef environment (Figure 1), manifesting as a dark-colored band separating healthy coral tissue from recently exposed coral skeleton. The band migrates horizontally across the surface of a coral colony, causing coral tissue necrosis at a rate of approximately 3.0 mm per day . The tissue loss on an individual colony infected with BBD can be substantial and can result in total colony mortality , , . Massive reef-building corals are susceptible to BBD , , , which exacerbates the impact of the disease on reef ecology and function . thumbnail Download: PPT PowerPoint slide PNG larger image (6.93MB) TIFF original image (5.69MB) Figure 1. Black band disease infection on a colony of Diploria strigosa on a reef of Curaçao. The dark-colored black band disease microbial mat separates apparently healthy coral tissue from white, denuded coral skeleton. Photograph provided by Abigael Brownell. doi:10.1371/journal.pone.0108541.g001 The BBD mat consists of a microbial consortium dominated by filamentous cyanobacteria , , , , , . Mechanisms of BBD pathogenicity include anoxic conditions within the BBD mat in combination with sulfide production by BBD sulfate reducing bacteria , and BBD cyanobacterial toxin (microcystin) production , , , . A recent microscopic study  documented cellular necrosis (i.e., loss of tissue confluence, cell-to-cell adhesion, cytoplasmic disintegration, nuclear breakdown, and the presence of autophagous bodies, pyknotic nuclei, and apoptotic bodies) in the coral tissue surrounding cyanobacterial filaments in active BBD infections. These microscopic observations support the previous studies demonstrating the role cyanobacterial toxins in BBD pathogenicity. A meta-analysis of 87 published BBD clone libraries from the Caribbean and Indo-Pacific  detected a common cyanobacterial sequence, recently characterized as Roseofilum reptotaenium gen. et sp. nov. , as present in 78% of the clone libraries. In contrast to the low diversity of BBD cyanobacterial taxa, the meta-analysis also revealed an extremely high diversity of heterotrophic BBD bacteria, with 73% of all sequences detected present as singletons (only 1 copy in the 87 clone libraries). Very little is known about the role of BBD heterotrophic bacteria, the one exception being that BBD has a well-documented and very active sulfur cycle generated by sulfate reducing bacteria within the disease consortium , . BBD infections occur on the surface tissues of infected coral colonies, which are also known to be microbially diverse. In particular, the coral surface mucopolysaccharide layer (SML) supports a dynamic microbial community ,  which is believed to play an important role in coral disease resistance . Interactions between several bacterial coral pathogens and the microbial community of the coral SML have been the subject of multiple studies (reviewed in ), and the coral SML and its associated microbes have been shown to produce antimicrobial agents and biofilm inhibitors that may be acting to protect corals from pathogen growth , , , , . However, relatively little is currently known about the interactions between microbes within the SML. Chemical signaling in tropical and subtropical coastal environments is a relatively unexplored area that may be important in coral health and disease. Quorum sensing (QS), a density dependent form of bacterial cell-cell communication, is one mechanism by which coral pathogenic bacteria and SML bacteria may be interacting . Acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2) are two of the more well-characterized groups of QS signaling molecules . The latter (AI-2) is a family of related molecules derived from (S)-4,5-dihydroxy-2,3-pentanedione (DPD) . AHLs are considered to be intraspecies signaling molecules , although it has been shown that cross-talk between bacteria using these molecules can occur (e.g., , , ). AHL production has been well documented among members of the proteobacteria , , a group that is commonly detected in BBD clone libraries , , , , , making AHL production a prime target for BBD research. AI-2 signaling is widely recognized as having an important role in interspecies communication , , , , since DPD production is common in both Gram-negative and Gram-positive bacteria , . AI-2 signaling may also play a role in the complex bacterial communities of both BBD and coral SML. Overall, QS is associated with a wide range of interactive social responses in bacteria (see , ) and has been shown to regulate virulence in both Gram-positive and Gram-negative pathogens , including upregulation of antibiotic biosynthesis , . AHL production has been observed in coral-associated bacteria isolated from the SML of healthy corals , isolates from other marine invertebrates and their endosymbiotic dinoflagellates , and from the tissues of 10 cnidarian species (all healthy) examined recently . Vibrio spp. isolated from the mucus of healthy and diseased corals have been shown to produce both AHLs and DPD . In one study, QS was proposed to play a major role in the pathogenicity of the coral pathogen Vibrio coralliitycus . With the exception of the study presented here, the in situ presence of QS signals in active coral disease has not yet been demonstrated. The roles of these signals in the coral microbial community and in coral disease remain unknown.