Temporal proteomic profiling reveals changes that support Burkholderia biofilms

Abstract

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.

Keywords: antimicrobial tolerance; biofilms; liquid chromatography mass spectrometry; melioidosis; proteomics.

Figure 1.

Antibiotic susceptibility evaluation of B. thailandensis to antimicrobial agents in planktonic and biofilm forms. The response of B. thailandensis to ceftazidime, ciprofloxacin, erythromycin and trimethoprim were shown (MIC; minimum inhibitory concentration, P-MIC; MIC of planktonic shedding cells and MBEC; minimum biofilm eliminating concentration).

Figure 2.

Principal component analysis (PCA) of biofilm and planktonic proteomics data (Log2 fold changes). The PCA plot represents 875 proteins with biological replicates that indicated clear proteomics profile differences between biofilm and planktonic states as in colors and matching symbols. PCA score plot was generated from planktonic (Early-exponential phase (E-log), Mid-exponential phase (M-log), Early-stationary phase (E-sta) and Mid-stationary phase (M-sta)) and biofilm sample (the early (B1D), maturing (B3D) and late (B6D) biofilm stages)

Figure 3.

Cluster of orthologous groups (COG) classification of 479 ANOVA significant and differentially abundant proteins with their classification (x-axis) and number of differentially regulated proteins (y-axis; both increased and decreased abundant). The total sum of COG classified proteins is greater than 479 proteins as some proteins fit in more than one COG classifiers: [R/S] Poorly characterized (R, S and uncharacterized); [X] Mobilome: prophages, transposons; [Q] Secondary metabolites biosynthesis, transport and catabolism; [P] Inorganic ion transport and metabolism; [I] Lipid transport and metabolism; [H] Coenzyme transport and metabolism; [G] Carbohydrate transport and metabolism; [F] Nucleotide transport and metabolism; [E] Amino acid transport and metabolism; [C] Energy production and conversion; [T] Signal transduction mechanisms; [U] Intracellular trafficking, secretion, and vesicular transport; [O] Posttranslational modification, protein turnover, chaperones; [N] Cell motility; [M] Cell wall/membrane/envelope biogenesis; [D] Cell cycle control, cell division, chromosome partitioning; [L] Replication, recombination and repair; [K] Transcription; [J] Translation, ribosomal structure and biogenesis.

Figure 4.

Protein abundance (z-scored) profiles of Burkholderia virulence factors during planktonic and biofilm growth. About 121 manually curated virulence factors and stress-related proteins are compared for their temporal abundance between planktonic and biofilm samples. Cluster (1), (2) and (3) show proteins, whose abundance is increased in Early-/Mid-log, Early/Mid-stationary and Early/Maturing/Late biofilm samples, respectively. Cluster (1) and (2) have majority of differentially regulated Burkholderia virulence factors and cluster (3) contains stress-related response proteins (BTH_I2608, BTH_I0164, BTH_I0498, BTH_I2122, BTH_I0763, BTH_I2418 and BTH_I2092) (Table S2, Supporting Information).

Figure 5.

Proteome rewiring supports bacterial biofilm.