Algal Auxins Decrease the Virulence of Vibrio Campbellii, a Major Pathogen of Aquatic Organisms

Environmental Microbiology (2017) 19(5), 1987–2004 doi:10.1111/1462-2920.13714 Indole signalling and (micro)algal auxins decrease the virulence of Vibrio campbellii, a major pathogen of aquatic organisms Qian Yang,1 Gde Sasmita Julyantoro Pande,1 indole-3-acetamide, which were produced by various Zheng Wang,2 Baochuan Lin,2† Robert A. Rubin,3 (micro)algae sharing the aquatic environment with Gary J. Vora2 and Tom Defoirdt1,4* V. campbellii, have a similar effect as observed for 1Laboratory of Aquaculture & Artemia Reference indole. Auxins might, therefore, have a significant Center, Ghent University, Gent, Belgium. impact on the interactions between vibrios, (micro)al- 2Center for Bio/Molecular Science & Engineering, Naval gae and higher organisms, with major ecological and Research Laboratory, Washington, DC, USA. practical implications. 3Mathematics Department, Whittier College, Whittier, CA, USA. 4Center for Microbial Ecology and Technology (cmet), Ghent University, Gent, Belgium. Introduction Vibrio campbellii and related species belonging to the Har- Summary veyi clade of vibrios, are marine Gram-negative bacteria that can infect a wide range of wild and cultured aquatic Vibrios belonging to the Harveyi clade are major organisms (both vertebrates and invertebrates), leading to pathogens of marine vertebrates and invertebrates, significant losses in the aquaculture industry worldwide causing major losses in wild and cultured organisms. (Austin and Zhang, 2006; Ruwandeepika et al., 2012; Despite their significant impact, the pathogenicity Dong et al., 2016). Despite their role as major pathogens mechanisms of these bacteria are not yet completely of marine animals, the pathogenicity mechanisms of these understood. In this study, the impact of indole signal- bacteria are not yet fully understood. The factors that have ling on the virulence of Vibrio campbellii was been considered to be involved in pathogenicity include investigated. Elevated indole levels significantly biofilm formation, swimming motility and the production of decreased motility, biofilm formation, exopolysac- a variety of extracellular products (Karunasagar et al., charide production and virulence to crustacean 1994; Austin and Zhang, 2006; Ruwandeepika et al., hosts. Indole furthermore inhibited the three-channel 2012; Yang and Defoirdt, 2015). quorum sensing system of V. campbellii, a regulatory Since virulence factors are often costly metabolic prod- mechanism that is required for full virulence of the ucts, their expression usually is under strict regulatory pathogen. Further, indole signalling was found to control. One of the regulatory mechanisms controlling the interact with the stress sigma factor RpoS. Together production of virulence factors in V. campbellii is quorum with the observations that energy-consuming pro- sensing, cell-to-cell communication with secreted signalling cesses (motility and bioluminescence) are molecules (Defoirdt et al., 2008). V. campbellii strain ATCC downregulated, and microarray-based transcriptom- BAA-1116 (previously designated as V. harveyi;Linet al., ics demonstrating that indole decreases the 2010) is a model bacterium in quorum sensing expression of genes involved in energy and amino research and has been found to contain a three-channel acid metabolism, the data suggest that indole is a quorum sensing system that uses three kinds of signalling starvation signal in V. campbellii. Finally, it was molecules: harveyi autoinducer 1 (HAI-1; Cao and Meigh- found that the auxins indole-3-acetic acid and en,1989), autoinducer 2 (AI-2; Chen et al., 2002) and cholerae autoinducer 1 (CAI-1; Higgins et al., 2007). Previ- Received 13 January, 2017; accepted 25 February, 2017. *For cor- ous work in our lab has shown that the three-channel respondence. E-mail [email protected]; Tel. 132 (0)9 264 quorum sensing system is required for full virulence of V. 59 76; Fax 132 (0)9 264 62 48. †Present address: Defense Threat Reduction Agency, Chemical and Biological Technologies, Fort campbellii toward different aquatic hosts (Defoirdt and Sor- Belvoir, VA, USA. geloos, 2012; Pande et al., 2013). VC 2017 Society for Applied Microbiology and John Wiley & Sons Ltd 1988 Q. Yang et al. In recent years, more compounds have been added to its ecological perspective, algal-bacterial signalling mecha- the arsenal of bacterial signalling molecules, and one of nisms (e.g., using indoles) are of significant interest to these compounds is indole (Lee and Lee, 2010). Indole is aquaculture as they might be manipulated to steer the produced by tryptophanase (TnaA), which reversibly con- microbial activity in aquaculture systems in order to verts tryptophan into indole, pyruvate and ammonia (Lee decrease the incidence of diseases (Natrah et al., 2014). et al., 2007). Although a large variety of both Gram- Given the ecological and economical importance of V. positive and Gram-negative bacteria (more than 85 spe- campbellii as a major pathogen of aquatic organisms, the cies including many pathogens) have long been known to previous reports documenting the impact of indole on produce indole, knowledge of its role as a signalling mole- virulence-related phenotypes and virulence of vibrios, and cule is relatively recent (Lee et al., 2015b). Some bacteria the potential impact of microalgal metabolites on this pro- that lack tryptophanase, and therefore do not produce cess, we sought to determine the impact of indole indole, for example, Pseudomonas aeruginosa,also signalling on the virulence of V. c amp be ll ii and to investi- respond to the presence of extracellular indole (Chu et al., gate whether the indole analogues indole-3-acetic acid 2012). Diverse biological responses to indole have recently and indole-3-acetamide induce a similar response as been revealed, including spore formation (Stamm et al., indole. 2005), drug resistance (Hirakawa et al., 2005), plasmid stability (Chant and Summers, 2007), persister formation Results (Vega et al., 2012), motility (Bansal et al., 2007), lipopoly- Impact of indole on the virulence of V. campbellii toward saccharide production (Han et al., 2011) and biofilm gnotobiotic brine shrimp larvae and conventionally formation (Mueller et al., 2009). Additionally, indole has reared giant river prawn larvae been reported to interfere with acylhomoserine lactone (AHL)-based quorum sensing in a number of Gram- To examine whether indole could decrease the virulence of negative bacteria including Acinetobacter oleivorans, Chro- wild type V. c amp be ll ii, we performed a standardized chal- mobacterium violaceum, Pseudomonas chlororaphis and lenge test with gnotobiotic brine shrimp larvae. The Serratia marcescens (Kim and Park, 2013; Hidalgo- addition of 50 and 100 mM of indole to the brine shrimp Romano et al., 2014). To date, however, a role for indole rearing water (single addition at the start of the experi- signalling in vibrios has only been documented in V. ch ol - ment) significantly increased the survival of the challenged erae and very recently in V. anguillarum.InV. cholerae larvae (Supporting Information Table S1). However, indole strain SIO, indole activated Vibrio polysaccharide (VPS) appeared to be toxic to the larvae at higher concentrations production and biofilm formation, whereas it decreased since significant mortality was observed in larvae (both motility (Mueller et al., 2009). In V. anguillarum strain challenged and unchallenged) exposed to 200 mMindole. NB10, by contrast, exopolysaccharide production was Therefore, in a second experiment, V. c amp be ll ii was incu- decreased by indole, whereas motility was not affected (Li bated in the presence of indole at 50, 100 and 200 lM, et al., 2014). Furthermore, in V. anguillarum,indole respectively, after which the cultures were washed to signalling and the stress sigma factor RpoS are intercon- remove the indole prior to inoculation into the brine shrimp nected as deletion of rpoS increased indole production rearing water. This way, we could eliminate any direct (Li et al., 2014). effect of indole to the larvae, enabling us to unambiguously Vibrios share the aquatic environment with (micro)algae, determine the impact of indole on (the virulence of) V. and both groups of organisms are known to interact with campbellii. A significantly increased survival of brine each other in various ways (ranging from mutualistic to shrimp larvae challenged with indole-pretreated V. ca mp - antagonistic) (Mayali and Azam, 2004; Amin et al., 2015). bellii was observed (when compared with larvae While these interactions are most likely coordinated by sig- challenged with untreated V. c amp be ll ii), indicating that nalling mechanisms between bacteria and algae, little is indole indeed decreases the virulence of V. ca mp be ll ii known with respect to signalling mechanisms that occur (Table 1). Importantly, all treatments received the same between algae and bacteria (Zhou et al., 2016). Interest- inoculum (105 cells per ml brine shrimp rearing water), and ingly, various (micro)algae produce indole analogues such there were no differences between treatments with respect as indole-3-acetic acid and indole-3-acetamide (Support- to the density of V. campbellii in the brine shrimp rearing ing Information Fig. S1) (Stirk et al., 2013; Lu and Xu, water after 1 and 2 days of challenge (Supporting Informa- 2015). Although several bacteria (predominantly plant- tion Fig. S2), indicating that all larvae had been exposed to associated bacteria and the human pathogens E. coli and

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