Quorum Quenching Strains Isolated from the Microbiota of Sea Anemones and Holothurians Attenuate Vibriocorallilyticus Virulence Factors and Reduce Mortality in Artemiasalina

Abstract

Interference with quorum-sensing (QS) intercellular communication systems by the enzymatic disruption of N-acylhomoserine lactones (AHLs) in Gram-negative bacteria has become a promising strategy to fight bacterial infections. In this study, seven strains previously isolated from marine invertebrates and selected for their ability to degrade C6 and C10-HSL, were identified as Acinetobacter junii, Ruegeria atlantica, Microbulbifer echini, Reinheimera aquimaris, and Pseudomonas sihuiensis. AHL-degrading activity against a wide range of synthetic AHLs were identified by using an agar well diffusion assay and Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 and VIR07 as biosensors. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis indicated that this activity was not due to an AHL lactonase. All the strains degraded Vibrio coralliilyticus AHLs in coculture experiments, while some strains reduced or abolished the production of virulence factors. In vivo assays showed that strains M3-111 and M3-127 reduced this pathogen's virulence and increased the survival rate of Artemia salina up to 3-fold, indicating its potential use for biotechnological purposes. To our knowledge, this is the first study to describe AHL-degrading activities in some of these marine species. These findings highlight that the microbiota associated with marine invertebrates constitute an important underexplored source of biological valuable compounds.

Keywords: N-acylhomoserine lactone; acylase; holothurians; marine bacteria; quorum quenching; sea anemones.

Figure 1

Detection of the remaining C10-HSL after incubation with the AHL-degrading strains at pH 7 and pH 2. (a) Diffusion agar-plate assay to detect C10-HSL using the biosensor strain C. violaceum VIR07. (b) HPLC-MS measurements of C10-HSL. Values are referred to as % remaining AHLs. Cell-free MB medium was used as negative control. Initial AHL concentration was 10 µM. This assay was repeated three times. Error bars show standard deviations. (c) Schematic description of the QQ reactions that may occur.

Figure 2

Detection of AHLs and phenotypes (enzymatic activities and motility) in the monocultures and co-cultures of AHL-degrading strains and the pathogen V. corallilyticus VibC-Oc-193. A. tumefaciens NTL4 (pZLR4) was used as biosensor strain to detect AHLs.

Figure 3

Survival rate of A. salina nauplii after 24 h, 48 h and 72 h of incubation with V. coralliilyticus VibC-Oc-193 in the presence or absence of AHL-degrading strains. Sterile filtered seawater (SFSW) was used as control. Different letters above the bars indicate that the values are significantly different (p < 0.05). The statistical analysis was carried through a Tukey’s Multiple Comparison Test.