Mucus; the untold story (for corals at least)

Serratia marcescens has been identified as the etiologic agent of white pox disease in the Caribbean Staghorn coral Acropora palmata. Acroporid serratiosis is one of the first cases of ‘reverse marine zoonosis’, where a known human enterobacterium disseminated via inadequately treated effluent water into surrounding coastal waters has become a novel marine pathogen. First reported in Florida in 1996 white pox disease has more than decimated the once dominant A. palmata population in the Florida keys, and has since spread across the Caribbean resulting in greater than 70 % losses of affected reefs.

The exact mechanism by which S. marcescens causes disease in A. palmata remains unknown. However, opportunistic pathogens must first colonise and/or penetrate the coral’s surface mucus layer (SML) if they are to cause disease. The SML of Acropora spp is composed of a glycoprotein polymer derived from zooxanthellae photosynthate, secreted by coral polyp mucocytes. The SML can support bacterial populations of up to 10⁸, and pathogens must compete with abundant coral commensals for available nutrients. Both pathogens and commensals employ the use of glycosidases (carbohydrate hydrolysing enzymes) to utilise the SML for growth.

Krediet et al. (2013), investigated the importance of inducible glycosidase activity for the growth of S. marcescens on A. palmata mucus. The authors also investigated the ability of commensal bacteria to inhibit carbohydrate hydrolysis enzymatic activities of S. marcescens and prevent swarming as a method of mucus colonisation.

A S. marcescens transposon mutant (strain CK2A4) deficient in glycosidase and other carbohydrate hydrolysis enzyme activities was generated by mariner transposon mutagenesis utilising E. coli. The competitive fitness of mutant (CK2A4) vs wild type (PDL100) was assessed by 1:1 co-culture at 10² cfu ml^-1 onto A. palmata mucus, high molecular weight fraction of coral mucus, and a Casamino acid/glycerol medium. The mutant was not competitively fit against the wild type on either of the coral mucus substrates, due to its reduced glycosidase enzyme activity. After 24 hours the wild type represented 95 % of the co-culture. However, on the Casamino acid/glycerol substrate (not able to be degraded by the glycosidases tested) the mutant held a slight competitive advantage over the wild type.

Culturable A. palmata mucus commensals were isolated from healthy colonies and identified through PCR amplification and 16S rRNA fragment sequencing. Commensals were co-cultured with PDL100 in carbohydrate rich medium to test for β-galactosidase inhibitory action. Six commensal strains were identified as having glycosidase inhibitory effects. A competitive fitness assay for S. marcescens vs glycosidase inhibiting commensal bacteria (n =6) was performed on high molecular weight fraction of coral mucus with a 1:1 ratio at 10⁴ cfu per ml^-1. The coral commensals reduced the growth of the pathogen by 10-100 fold (compared to S. marcescens monoculture), and increased the fitness of the glycosidase deficient mutant, demonstrating that inhibition of glycosidases is at least partly responsible for the reduction in growth of the wild type. Commensal strain Exiguobacterium sp. 33G8 significantly inhibited S. marcescens glycosidase activity in co-culture and the inhibitory compound was further characterised as an extracellular, small molecular weight, ethanol-soluble compound. I was disappointed that the authors did not characterise the inhibitory compound produced by Exiguobacterium sp. 33G8 further, as it would be informative to discover if it was an antimicrobial, secondary metabolite etc.

5 of the 6 commensal bacteria were able to inhibit swarming of S. marcsecens (PDL100). The commensal strains were individually assayed by inoculating them upon a filter disc and testing for S. marcescens growth around it on AB swarm agar. Interestingly, commensal strain Exiguobacterium sp. 33G8 did not inhibit swarming although it had the highest glucosidase enzymatic inhibitory effect upon S. marcescens.

This study effectively demonstrates that glycosidases and swarming activity are important mechanisms for growth and successful colonisation of A. palmata’s SML by S. marcescens, and that selected commensal bacteria may play a protective role against opportunistic invaders. However, the commensals in this study were unable to prevent colonisation of the SML, only slow down the invasion. Importantly, some commensals that were effective in reducing enzyme activity were not effective against swarming by the pathogen and vice versa. This lends support to the probiotic hypothesis; that having a diverse, beneficial commensal community acts synergistically to protect the health of the host. Lastly, I did not agree that the sea anemone virulence model (not detailed here) was particularly relevant to the coral species at hand especially considering the unrealistically high inoculum levels chosen.

Reference:

Krediet, C. J., Ritchie, K. B., Alagely, A., & Teplitski, M. (2013). Members of native coral microbiota inhibit glycosidases and thwart colonization of coral mucus by an opportunistic pathogen. The ISME journal, 7(5), 980-990.