Cory J. Krediet

Coral-associated micro-organisms and their roles in promoting coral health and thwarting diseases

Over the last decade, significant advances have been made in characterization of the coral microbiota. Shifts in its composition often correlate with the appearance of signs of diseases and/or bleaching, thus suggesting a link between microbes, coral health and stability of reef ecosystems. The understanding of interactions in coral-associated microbiota is informed by the on-going characterization of other microbiomes, which suggest that metabolic pathways and functional capabilities define the ‘core’ microbiota more accurately than the taxonomic diversity of its members. Consistent with this hypothesis, there does not appear to be a consensus on the specificity in the interactions of corals with microbial commensals, even though recent studies report potentially beneficial functions of the coral-associated bacteria. They cycle sulphur, fix nitrogen, produce antimicrobial compounds, inhibit cell-to-cell signalling and disrupt virulence in opportunistic pathogens. While their beneficial functions have been documented, it is not certain whether or how these microbes are selected by the hosts. Therefore, understanding the role of innate immunity, signal and nutrient exchange in the establishment of coral microbiota and in controlling its functions will probably reveal ancient, evolutionarily conserved mechanisms that dictate the outcomes of host–microbial interactions, and impact the resilience of the host.

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.

Members of native coral microbiota inhibit glycosidases and thwart colonization of coral mucus by an opportunistic pathogen

The outcome of the interactions between native commensal microorganisms and opportunistic pathogens is crucial to the health of the coral holobiont. During the establishment within the coral surface mucus layer, opportunistic pathogens, including a white pox pathogen Serratia marcescens PDL100, compete with native bacteria for available nutrients. Both commensals and pathogens employ glycosidases and N-acetyl-glucosaminidase to utilize components of coral mucus. This study tested the hypothesis that specific glycosidases were critical for the growth of S. marcescens on mucus and that their inhibition by native coral microbiota reduces fitness of the pathogen. Consistent with this hypothesis, a S. marcescens transposon mutant with reduced glycosidase and N-acetyl-glucosaminidase activities was unable to compete with the wild type on the mucus of the host coral Acropora palmata, although it was at least as competitive as the wild type on a minimal medium with glycerol and casamino acids. Virulence of the mutant was modestly reduced in the Aiptasia model. A survey revealed that ∼8% of culturable coral commensal bacteria have the ability to inhibit glycosidases in the pathogen. A small molecular weight, ethanol-soluble substance(s) produced by the coral commensal Exiguobacterium sp. was capable of the inhibition of the induction of catabolic enzymes in S. marcescens. This inhibition was in part responsible for the 10–100-fold reduction in the ability of the pathogen to grow on coral mucus. These results provide insight into potential mechanisms of commensal interference with early colonization and infection behaviors in opportunistic pathogens and highlight an important function for the native microbiota in coral health.

Utilization of Mucus from the Coral Acropora palmata by the Pathogen Serratia marcescens and by Environmental and Coral Commensal Bacteria

ABSTRACT In recent years, diseases of corals caused by opportunistic pathogens have become widespread. How opportunistic pathogens establish on coral surfaces, interact with native microbiota, and cause disease is not yet clear. This study compared the utilization of coral mucus by coral-associated commensal bacteria (“Photobacterium mandapamensis” and Halomonas meridiana) and by opportunistic Serratia marcescens pathogens. S. marcescens PDL100 (a pathogen associated with white pox disease of Acroporid corals) grew to higher population densities on components of mucus from the host coral. In an in vitro coculture on mucus from Acropora palmata, S. marcescens PDL100 isolates outgrew coral isolates. The white pox pathogen did not differ from other bacteria in growth on mucus from a nonhost coral, Montastraea faveolata. The ability of S. marcescens to cause disease in acroporid corals may be due, at least in part, to the ability of strain PDL100 to build to higher population numbers within the mucus surface layer of its acroporid host. During growth on mucus from A. palmata, similar glycosidase activities were present in coral commensal bacteria, in S. marcescens PDL100, and in environmental and human isolates of S. marcescens. The temporal regulation of these activities during growth on mucus, however, was distinct in the isolates. During early stages of growth on mucus, enzymatic activities in S. marcescens PDL100 were most similar to those in coral commensals. After overnight incubation on mucus, enzymatic activities in a white pox pathogen were most similar to those in pathogenic Serratia strains isolated from human mucosal surfaces.

Catabolite regulation of enzymatic activities in a white pox pathogen and commensal bacteria during growth on mucus polymers from the coral Acropora palmata.

Colonization of host mucus surfaces is one of the first steps in the establishment of coral-associated microbial communities. Coral mucus contains a sulfated glycoprotein (in which oligosaccharide decorations are connected to the polypeptide backbone by a mannose residue) and molecules that result from its degradation. Mucus is utilized as a growth substrate by commensal and pathogenic organisms. Two representative coral commensals, Photobacterium mandapamensis and Halomonas meridiana, differed from a white pox pathogen Serratia marcescens PDL100 in the pattern with which they utilized mucus polymers of Acropora palmata. Incubation with the mucus polymer increased mannopyranosidase activity in S. marcescens, suggestive of its ability to cleave off oligosaccharide side chains. With the exception of glucosidase and N-acetyl galactosaminidase, glycosidases in S. marcescens were subject to catabolite regulation by galactose, glucose, arabinose, mannose and N-acetyl-glucosamine. In commensal P. mandapamensis, at least 10 glycosidases were modestly induced during incubation on coral mucus. Galactose, arabinose, mannose, but not glucose or N-acetyl-glucosamine had a repressive effect on glycosidases in P. mandapamensis. Incubation with the mucus polymers upregulated 3 enzymatic activities in H. meridiana; glucose and galactose appear to be the preferred carbon source in this bacterium. Although all these bacteria were capable of producing the same glycosidases, the differences in the preferred carbon sources and patterns of enzymatic activities induced during growth on the mucus polymer in the presence of these carbon sources suggest that to establish themselves within the coral mucus surface layer commensals and pathogens rely on different enzymatic activities.

Role of GacA in virulence of Vibrio vulnificus.

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.

N‐ACYL HOMOSERINE LACTONe LACTONASE, AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.

Outcomes of infections of sea anemone Aiptasia pallida with Vibrio spp. pathogenic to corals.

Incidents of coral disease are on the rise. However, in the absence of a surrogate animal host, understanding of the interactions between coral pathogens and their hosts remains relatively limited, compared to other pathosystems of similar global importance. A tropical sea anemone, Aiptasia pallida, has been investigated as a surrogate model to study certain aspects of coral biology. Therefore, to test whether the utility of this surrogate model can be extended to study coral diseases, in the present study, we tested its susceptibility to common coral pathogens (Vibrio coralliilyticus and Vibrio shiloi) as well as polymicrobial consortia recovered from the Caribbean Yellow Band Disease (CYBD) lesions. A. pallida was susceptible to each of the tested pathogens. A. pallida responded to the pathogens with darkening of the tissues (associated with an increased melanization) and retraction of tentacles, followed by complete disintegration of polyp tissues. Loss of zooxanthellae was not observed; however, the disease progression pattern is consistent with the behavior of necrotizing pathogens. Virulence of some coral pathogens in Aiptasia was paralleled with their glycosidase activities.

Interactions between the tropical sea anemone Aiptasia pallida and Serratia marcescens, an opportunistic pathogen of corals

Coral reefs are under increasing stress caused by global and local environmental changes, which are thought to increase the susceptibility of corals to opportunistic pathogens. In the absence of an easily culturable model animal, the understanding of the mechanisms of disease progression in corals remains fairly limited. In the present study, we tested the susceptibility of the tropical sea anemone Aiptasia pallida to an opportunistic coral pathogen (Serratia marcescens). A. pallida was susceptible to S. marcescens PDL100 and responded to this opportunistic coral pathogen with darkening of the tissues and retraction of tentacles, followed by complete disintegration of polyp tissues. Histological observations revealed loss of zooxanthellae and structural changes in eosinophilic granular cells in response to pathogen infection. A screen of S. marcescens mutants identified a motility and tetrathionate reductase mutants as defective in virulence in the A. pallida infection model. In co-infections with the wild-type strain, the tetrathionate reductase mutant was less fit within the surface mucopolysaccharide layer of the host coral Acropora palmata.

Microbial Interactions on Coral Surfaces and Within the Coral Holobiont

Microbial communities associated with coral surfaces are diverse and complex. They play key roles in nutrient acquisition by coral holobionts and in responses to stressors and diseases. Members of coral-associated microbiota produce antimicrobial compounds, inhibit cell-to-cell signaling, and disrupt virulence in opportunistic pathogens. Characterization of coral-associated microbial communities suggests that metabolic capabilities define the core members of the communities. However, some taxonomic conservation is becoming evident in microbial communities associated with the same coral species and genera in different geographic regions. Even though shifts in the composition of coral microbiota often correlate with the appearance of signs of diseases and/or bleaching, it is not yet clear to what extent these shifts are a cause or a consequence of diseases. This chapter focuses on interactions within coral-associated microbial communities and suggests potentially interesting directions for future research.