Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

Abstract

Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm.

Non-hierarchical, RhlR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate ofPseudomonas aeruginosa

“…Moreover, we observed that the RhlR regulon included genes that likely confer a growth advantage in the CF lung. For example, cbpD encodes a chitin-binding protein shown to contribute to the thickness of biofilms, the development of which is important for nutrient acquisition and stress resistance [36]. The gene encoding the monodechloroaminopyrrolnitrin 3-halogenase PrnC was also present in the E90 RhlR regulon, which has not been reported in previous P. aeruginosa transcriptomes and is not present in the PAO1 reference genome.…”

EPS—Then and Now

“…An interesting example of how biofilm organisms practice self-organization is offered by Bacillus subtilis. When growth slows due to nutrient depletion, nearly all bacteria are encased in the matrix, which is triggered by nutrient depletion [93], and the mature biofilm looks macroscopically wrinkled. In the late stages of B. subtilis biofilm development, severe starvation occurs and hundreds of cells pile up in aerial structures known as fruiting bodies that serve as preferential sites for sporulation [89].…”

Critical review on biofilm methods

“…The protein fraction of extracted EPS is usually obtained by trichloroacetic acid (TCA) precipitation while the carbohydrate fraction is purified and concentrated by ethanol precipitation (Jiao et al, 2010). Then, carbohydrates could be characterized using multiple analytical techniques from hydrolyzed polysaccharides to obtain a carbohydrate fingerprint of biofilm EPS while protein diversity could be explored using proteomics approaches (Gallaher et al, 2006;Lilledahl & Stokke, 2015;Speziale et al, 2014;Zhang et al, 2015b).…”