Lactonase-mediated inhibition of quorum sensing largely alters phenotypes, proteome, and antimicrobial activities in Burkholderia thailandensis E264

Abstract

Introduction: Burkholderia thailandensis is a study model for Burkholderia pseudomallei, a highly virulent pathogen, known to be the causative agent of melioidosis and a potential bioterrorism agent. These two bacteria use an (acyl-homoserine lactone) AHL-mediated quorum sensing (QS) system to regulate different behaviors including biofilm formation, secondary metabolite productions, and motility.

Methods: Using an enzyme-based quorum quenching (QQ) strategy, with the lactonase SsoPox having the best activity on B. thailandensis AHLs, we evaluated the importance of QS in B. thailandensis by combining proteomic and phenotypic analyses.

Results: We demonstrated that QS disruption largely affects overall bacterial behavior including motility, proteolytic activity, and antimicrobial molecule production. We further showed that QQ treatment drastically decreases B. thailandensis bactericidal activity against two bacteria (Chromobacterium violaceum and Staphylococcus aureus), while a spectacular increase in antifungal activity was observed against fungi and yeast (Aspergillus niger, Fusarium graminearum and Saccharomyces cerevisiae).

Discussion: This study provides evidence that QS is of prime interest when it comes to understanding the virulence of Burkholderia species and developing alternative treatments.

Keywords: Acyl-homoserine lactone; Burkholderia thailandensis; lactonase; quorum quenching; quorum sensing.

Figure 1

AHLs detection in cell-free extracts of B. thailandensis. AHL detection in B. thailandensis cell-free extracts using an E. coli MT102 reporter strain after cultivation without and with SsoPox V82I (0.5 mg.mL^-1). C₈-HSL, 3-OH-C₈-HSL and 3-OH-C₁₀-HSL were used as a positive control at 100 µM. Error bars represent standard deviations for n=3 biological replicates. **p-value < 0.01 according to Student’s t-test.

Figure 2

SsoPox V82I disrupts biofilm formation and aggregation in B. thailandensis. (A) Mean levels of biofilm formation by B. thailandensis without enzymatic treatment (gray) and treated with 0.5 mg.mL^-1 SsoPox V82I (yellow). Error bars represent the standard deviations of n=4 biological replicates. ***p-value < 0.001 according to Student’s t-test. Representative pictures of crystal violet staining of biofilm formation are presented below each condition. (B) Cell aggregation in shaken culture treated with SsoPox V82I (0.5 mg.mL^-1) or untreated after 16 hours culture at 37°C.

Figure 3

B. thailandensis E264 proteome changes upon SsoPox V82I treatment. (A) PLS-DA analysis of proteomic analysis of n=4 biological replicates. Untreated samples are represented in gray and samples treated with SsoPox V82I (0.5 mg.mL^-1) are in yellow. (B) Table summarizing the number of proteins impacted by SsoPox V82I treatment and their respective proportion to the B. thailandensis proteome or number of significantly impacted proteins. (C) Representation of proteomic fold changes as a violin plot. Logarithmic fold changes of 2,495 proteins are plotted and proteins are classified according to their functions. The central line represents a null fold change, meaning no modification occurs with SsoPox V82I treatment. The x=0.58 and x=-0.58 lines correspond respectively to a 50% increase or decrease.

Figure 4

QS disruption in B. thailandensis increases proteolytic activity and motility. (A) Heatmap representing the fold changes of protease proteins between untreated conditions and those treated with SsoPox V82I (0.5 mg.mL^-1). (B) Mean levels of B. thailandensis proteolytic activity on skimmed milk agar plates as measured with ImageJ software of treated SsoPox V82I (0.5 mg.mL^-1) and untreated conditions. Error bars represent the standard deviations of n=3 biological replicates. ***p-value < 0.001 according to Student’s t-test. Pictures of proteolytic halos are presented below each condition. (C) Heatmap representing the fold changes of proteins involved in motility between untreated and treated SsoPox V82I (0.5 mg.mL^-1) conditions. (D) Modulation of swarming motility in B. thailandensis after lactonase treatment with SsoPox V82I (0.5 mg.mL^-1) on 0.7% agar plates. Error bars represent the standard deviations of n=6 biological replicates. ***p-value <0.001 according to Student’s t-test.

Figure 5

SsoPox V82I treatment impacts T6SS protein levels in B. thailandensis. Heatmap of fold-changes in proteins related to T6SS between cells grown in the presence of SsoPox V82I (0.5 mg.mL^-1) and untreated cells.

Figure 6

Impact of enzymatic treatment on antimicrobial production. Heatmap of fold-changes of proteins involved in antimicrobial molecule production between cells grown in the presence of SsoPox V82I (0.5 mg.mL^-1) and untreated cells.

Figure 7

Decrease of bactericidal effect of B. thailandensis cell-free supernatants upon QQ. Twenty four hours of bacterial growth kinetics measured by OD 600 nm of (A) C. violaceum and (B) S. aureus in the presence of 50% of B. thailandensis supernatants obtained from cultures untreated or treated with SsoPox V82I (0.5 mg.mL^-1). Error bars represent the standard deviations of n= 4 biological replicates for control growth curves without supernatant (in violet and green) and n=6 biological replicates for conditions supplemented with supernatant (in yellow and gray).

Figure 8

Lactonase treatment increases the antifungal effect of B. thailandensis supernatants (A) Representative pictures of fungal growth in the presence of B. thailandensis cell-free supernatants obtained from cultures treated with SsoPox V82I (0.5 mg.mL^-1) or not on A. niger and F. graminearum. Pictures taken after 96 hours of growth at 25°C. (B) Effect of B. thailandensis cell-free supernatants obtained from cultures treated with SsoPox V82I (0.5 mg.mL^-1) or untreated against S. cerevisiae. Growth was followed by measuring OD at 600 nm for 24 hours. Error bars represent the standard deviations of n= 6 biological replicates. Pictures of S. cerevisiae colonies recovered from liquid cultivation are presented next to each condition.

Figure 9

SsoPox treatment decreases the abundance of bactobolins in bacterial supernatants. (A) Structures of bactobolins detected in this study. (B) Signal detection in LC-MS analysis of several bactobolin molecules after extraction with ethyl acetate of B. thailandensis supernatants either untreated or treated with SsoPox V82I (0.5 mg.mL^-1).