Pseudomonas chlororaphis PA23 metabolites protect against protozoan grazing by the predator Acanthamoeba castellanii

BACKGROUND: Pseudomonas chlororaphis strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator Acanthamoeba castellanii (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction. METHODS: Ac was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses. RESULTS: PRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 phzI/phzR quorum-sensing (QS) genes and phzA and prnA, which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly. CONCLUSIONS: PA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.