Piscirickettsia salmonis Produces a N-Acetyl-L-Homoserine Lactone as a Bacterial Quorum Sensing System-Related Molecule

Abstract

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, the most prevalent disease in salmonid species in Chilean salmonids farms. Many bacteria produce N-acyl-homoserine lactones (AHLs) as a quorum-sensing signal molecule to regulate gene expression in a cell density-dependent manner, and thus modulate physiological characteristics and several bacterial mechanisms. In this study, a fluorescent biosensor system method and gas chromatography-tandem mass spectrometry (GC/MS) were combined to detect AHLs produced by P. salmonis. These analyses revealed an emitted fluorescence signal when the biosensor P. putida EL106 (RPL4cep) was co-cultured with both, P. salmonis LF-89 type strain and an EM-90-like strain Ps007, respectively. Furthermore, the production of an AHL-type molecule was confirmed by GC/MS by both P. salmonis strains, which identified the presence of a N-acetyl-L-homoserine Lactone in the supernatant extract. However, It is suggested that an alternate pathway could synthesizes AHLs, which should be address in future experiments in order to elucidate this important bacterial process. To the best of our knowledge, the present report is the first to describe the type of AHLs produced by P. salmonis.

Keywords: AHL (N-acyl-homoserine lactone); N-acetyl-L-homoserine lactone; Piscirickettsia salmonis; SRS; quorum sensing (QS).

Figure 1

Selection of biosensor strains at the optimal growth conditions of P. salmonis. (A) Response of 4 μM C8-HSL to biosensor strains; PP-22: P. putida EL105 (RPL4las), PP-23: P. putida EL106 (RPL4cep), and EC-12: E. coli MT102 (pKR-C12) at 27°C for 24 h. Control: DMSO (C8-HSL solvent). (B) Response of 250 nM C8-HSL to P. putida EL 106 (RPL4cep) at 18°C and 27°C for 24 h. Emitted fluorescence was recorded in a microtiter plate reader at 515 nm. RFU, relative fluorescence units. Asterisks indicate statistical significance (Student’s t-test P ≤ 0.01, n = 3). Error bars: standard deviation.

Figure 2

Fluorescence induction in P. putida EL 106 (RPL4cep) in co-culture with P. salmonis LF-89^T and EM-90-like (Ps007) strains. (A) P. putida EL 106 (RPL4cep) 10⁵ cell/mL and 10⁹ cell/mL at 24, 96, 144 and 168h. Asterisks indicate statistical significance (ANOVA P ≤ 0.01, n = 3) in P. salmonis Ps007 strain between 10⁵ and 10⁹ cells/mL of the biosensor strain from 96 h to 168 h. In P. salmonis LF-89 strain asterisks indicate statistical significance (ANOVA P ≤ 0.01, n = 3) between 10⁵ and 10⁹ cells/mL of the biosensor strain only at 96 h. (B) P. putida EL 106 (RPL4cep) 10⁵ cell/mL at 120 h. Control: P. putida EL106 (RPL4cep) + 250 nM C8-HSL. Emitted fluorescence was recorded in a microtiter plate reader at 515 nm. RFU, relative fluorescence units. Asterisks indicate statistical significance (ANOVA P ≤ 0.01, n = 3) with the control. Error bars: standard deviation.

Figure 3

Fluorescence induction in P. putida EL 106 (RPL4cep) of supernatants and organic extracts of P. salmonis at different times. The filtered supernatant and organic extracts (dissolved in 10% w/v ASB) of each P. salmonis strain were arranged in a 1:1 ratio with the bacterial biosensor P. putida EL106 (RPL4cep) (10⁹ cells/mL). Samples and controls were incubated for 24 and 48 h at 27°C. PP23: P. putida EL 106 (RPL4cep); Controls: P. putida EL106 (RPL4cep) (10⁹ cells/mL) + 250 nM C8-HSL. Emitted fluorescence was recorded in a microtiter plate reader at 515 nm. RFU, relative fluorescence units. Asterisks indicate statistical significance (ANOVA P ≤ 0.01, n = 3) between the supernatant and the organic extracts. Error bars: standard deviation.

Figure 4

Identification of AHLs molecules in P. salmonis culture organic extract by GC/MS analysis. LF-89^T (A) and Ps007 (B) spectrum. Mass spectrum at t=10.459 min was included for both samples. The standard solutions of C4-HSL, C6-HSL, C8-HSL, and C12-HSL were prepared in GC-ECD/FID-grade acetone. MS conditions were as follows: electron ionization source set to 70 eV, MS Quad 150°C, MS Source 230°C. The mass spectrometer was run in full-scan mode (m/z 15–350) and Selected Ion Monitoring (SIM) at m/z 143.The sensor and GC/MS results are representative of three independent experiments.