AHL-Lactonase Producing Psychrobacter sp. From Palk Bay Sediment Mitigates Quorum Sensing-Mediated Virulence Production in Gram Negative Bacterial Pathogens

Abstract

Quorum sensing (QS) is a signaling mechanism governed by bacteria used to converse at inter- and intra-species levels through small self-produced chemicals called N-acylhomoserine lactones (AHLs). Through QS, bacteria regulate and organize the virulence factors' production, including biofilm formation. AHLs can be degraded by an action called quorum quenching (QQ) and hence QQ strategy can effectively be employed to combat biofilm-associated bacterial pathogenesis. The present study aimed to identify novel bacterial species with QQ potential. Screening of Palk Bay marine sediment bacteria for QQ activity ended up with the identification of marine bacterial isolate 28 (MSB-28), which exhibited a profound QQ activity against QS biomarker strain Chromobacterium violaceum ATCC 12472. The isolate MSB-28 was identified as Psychrobacter sp. through 16S-rRNA sequencing. Psychrobacter sp. also demonstrated a pronounced activity in controlling the biofilm formation in different bacteria and biofilm-associated virulence factors' production in P. aeruginosa PAO1. Solvent extraction, heat inactivation, and proteinase K treatment assays clearly evidence the enzymatic nature of the bioactive lead. Furthermore, AHL's lactone ring cleavage was confirmed with experiments including ring closure assay and chromatographic analysis, and thus the AHL-lactonase enzyme production in Psychrobacter sp. To conclude, this is the first report stating the AHL-lactonase mediated QQ activity from marine sediment bacteria Psychrobacter sp. Future work deals with the characterization, purification, and mass cultivation of the purified protein and should pave the way to assessing the feasibility of the identified protein in controlling QS and biofilm-mediated multidrug resistant bacterial infections in mono or multi-species conditions.

Keywords: AHL-lactonase; Pseudomonas aeruginosa; Psychrobacter sp.; marine Palk Bay sediment; quorum quenching.

Figure 1

Confocal microscopy images of bacterial biofilms harbored with and without Psychrobacter sp. CFS (200 μl/ml). Images represent the untreated controls (insert-C) and Psychrobacter sp. treated (insert-T) biofilms of PAO1 (A), S. marcescens (B), V. vulnificus (C), and V. parahaemolyticus (D).

Figure 2

Effect of Psychrobacter sp. on virulence factors production in PAO1. (A) Rhamnolipd production in PAO1. Image represents rhamnolipid production in untreated control (1), and PAO1 treated with CFS of Psychrobacter sp. at of 100 (2) and 200 μl/ml (3) Concentrations. (B) Assessment of biosurfactant production through drop collapse assay. Images represent the activity of untreated PAO1 (a), and PAO1 treated with CFS of Psychrobacter sp. at concentrations of 50–200 μl/ml (b-e). (C) Quantitative analysis of EPS inhibition in PAO1 cells treated with and without Psychrobacter sp. CFS. Data represent the OD values of the EPS. Mean values represent the data of three independent experiments and SD are shown. (D) Effect of Psychrobacter sp. on the motility of PAO1. Swimming and swarming motilities of untreated control of PAO1 (a,c) and PAO1 treated with Psychrobacter sp. CFS (200 μg/ml; b,d), respectively. (E) Analysis of PAO1 biofilm formation in the absence (a) and presence (b) of Psychrobacter sp. through ring assay.

Figure 3

Effect of solvents (A), heat treatment (B), and proteinase K (C) on QQ activity of Psychrobacter sp.

Figure 4

Analysis of C₆HSL degradation by Psychrobacter sp. through TLC method. (A) C₆HSL alone. (B) C₆HSL treated with CFS of Psychrobacter sp. at different concentrations.

Figure 5

Analysis of C₆HSL degradation by Psychrobacter sp. through HPLC. (A) C₆HSL alone. (B) C₆HSL treated with Psychrobacter sp.

Figure 6

(A) Analysis of mechanism of action of Psychrobacter sp. by ring closure assay. (B) Activity after HCL treatment. C₆HSL alone (a), degradation pattern of C₆HSL by CFS of Psychrobacter sp., (b) PAO1 (c), and B. subtilis (d).