Recent research has suggested that many emerging diseases are caused by opportunistic pathogens derived from populations present in the environment. An underlying assumption of this hypothesis is that non-pathogenic microbes harbor the genetic potential to become pathogenic if the opportunity arises. This project will address this question by comparing, using metabolomics and metagenetics, two cyanobacterial-dominated microbial mat communities, one that is a pathogenic biofilm and one that is based on autotrophy in an extreme environment. Microbial communities in extreme environments are considered to be primitive in an evolutionary context and have been studied intensively as analogues to early life on Earth. Microbial mats in a hot spring contain the same major microorganisms to the genus level as the pathogenic microbial consortium known as black band disease (BBD) of corals. Furthermore, the overall physicochemical structure and major physiological organization of the two systems are identical. Despite the identical taxonomic/physiological structure of the two populations, they are very different in terms of trophic structure. The hot-spring mats are photoautotrophic with two energy sources: light, which powers cyanobacterial photosynthesis, and sulfide dissolved in the geothermal source waters, which powers sulfur chemolithotrophy. The BBD mats, which also contain photosynthesizing cyanobacteria, are fueled by organic carbon (the coral host tissue) as well as sulfide produced by sulfate-reducing BBD bacteria that, in turn, grow on coral-derived organic carbon. This project will use an evolutionary context and comparative approach to construct metabolic networks for both systems based on complementary metabolomics and metagenetics, a comparison of the genetic potential (genomics data) with targeted metabolites (metabolomics data) and an assessment of differences in metabolites between the ancient photoautotrophic based community and the modern pathogenic community.
The health of coral reefs is now of global concern. This study will enhance existing knowledge by investigating the underlying potential for natural microbial communities to evolve pathogenicity. Research will include training of minority and female undergraduate and graduate students who will assist in the proposed research. All participating institutions are active in the recruitment and training of underrepresented groups. Florida International University is a doctoral-degree granting minority institution that is ranked first in the US among four year colleges for awarding bachelor's and master's degrees to Hispanic students, and first in awarding degrees to underrepresented minorities in STEM disciplines. The results of this study will be presented at national and international meetings, including the biannual scientific meetings of the Association of Marine Laboratories of the Caribbean (AMLC). The AMLC has science/education representatives and members from over 30 Caribbean/Latin American countries.
This project involved a comparison of two seemingly very different microbial communties - a microbial mat in a hot spring outflow in Oregon, and a complex pathogenic disease of corals called black band disease (BBD). Both communites are dominated by filamentous cyanobacteria that form dense mats that contain numerous heterotrophic bacteria and bacteria associated with the sulfur biogeochemical cycle. Both contain high levels of sulfide, entering the system from geothermal source waters in the hot springs and by sulfidogenic (sulfate reducing) bacteria in BBD. In both systems the cyanobacteria produce a potent cyanotoxin, microcystin. The hot spring microbial system is non pathogenic and based on photosynthesis, whereas the BBD system is pathogenic and fueled by lysing coral tissue killed by the activity of the band. The similarities of the system in terms of biological structure, sulfur biogeochemistry and toxins make them ideal for a comparative study of the evolution of pathogenicity. We found that production of the cyanotoxin is controlled in the pathogenic BBD by temperature, which is in accord with the overall disease dynamics in that the disease does not appear on coral reefs until temperature increases above 27.5 C. This is the temperature when the host coral begins to be stressed, and the BBD cyanobacteria produce increasing amounts of cyanotoxin as the temperature rises. pH does not affect microcystin production in BBD, which again is important in terms of pathogencity since there are wide changes in pH in the band on a daily basis due to the effects of photosynthesis (consumes CO2) and respiration (produces CO2) by BBD community members. In the hot spring system both temperature and pH affect microcystin production, causing overall decrease in production when levels move away (above or below) temperature and pH optima. This system is not pathogenic, and since production of the toxin is energy-consuming it is efficient to limit production to energy sufficient periods. It has been proposed that microcystin evolved as a cell signalling molecule which would explain why it is present in the non pathogenic hot spring system. BBD is a highly complex polymicrobial disease found on reefs worldwide. In this project we have sampled and compared BBD on reefs of the Florida Keys and the southern Caribbean Sea (Curacao) to investigate mechanisms of pathogenicity. We have detected and quantified different quorum sensing (QS) signalling molecules that are known, in general, to regulate virulence in different diseases. These QS molecules are produced by heterotrophic bacteria that live in the band, and their production is, in some cases, controlled by temperature. We are in the process of mining metagenomic data produced in this study to determine which virulence/quorum sensing genes are present in both communities and their co-occurrence. Since microcystin is also implicated in cell signalling we investigated this potential role within BBD. We found that exposure of the BBD community to purified microcystin changes production of metabolites, and in particular promotes pathways involved in nucleic acid biosynthesis, resulting in upregulation of the pathways. Thus this work has corroborated the hypothesis that microcystin evolved as a cell signalling molecule, and that microcystin only secondarily evolved as a toxin. We are still analyzing metagenomics and metabolomics data sets produced within the project to understand the interconnected processes of toxin activity and production, cell signalling, and physiology of the bacterial components of the two systems. This project is of importance in terms of the observed worldwide decline in coral reef health, and in the increasing cases of toxic blooms of cyanobacteria. Coral disease is believed to be one of the major factors involved in global coral reef decline, and black band disease is one of the most destructive and complex of these diseases. Our results have helped to further understand the pathobiology of this disease, and will be of importance to reef managers. Our results on the relationships between environmental factors and microcystin production will be useful in combating and controlling the increasingly common blooms of toxic cyanobacteria in both fresh waters and marine coastal zones. This project has supported three female graduate students and one female post-doctoral associate. In addition to publishing results in peer-reviewed journals we have made numerous presentations at local, national, and international conferences. In particular this work has been presented at the Scientific Meetings of the Association of Marine Laboratories of the Caribbean, an international meeting attended by scientists, students, researchers and managers from many countries throughout the wider Caribbean.
