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Single microcolony diffusion analysis in Pseudomonas aeruginosa biofilms.

ABSTRACT: The influence of the biofilm matrix on molecular diffusion is commonly hypothesized to be responsible for emergent characteristics of biofilms such as nutrient trapping, signal accumulation and antibiotic tolerance. Hence quantifying the molecular diffusion coefficient is important to determine whether there is an influence of biofilm microenvironment on the mobility of molecules. Here, we use single plane illumination microscopy fluorescence correlation spectroscopy (SPIM-FCS) to obtain 3D diffusion coefficient maps with micrometre spatial and millisecond temporal resolution of entire Pseudomonas aeruginosa microcolonies. We probed how molecular properties such as size and charge as well as biofilm properties such as microcolony size and depth influence diffusion of fluorescently labelled dextrans inside biofilms. The 2?MDa dextran showed uneven penetration and a reduction in diffusion coefficient suggesting that the biofilm acts as a molecular sieve. Its diffusion coefficient was negatively correlated with the size of the microcolony. Positively charged dextran molecules and positively charged antibiotic tobramycin preferentially partitioned into the biofilm and remained mobile inside the microcolony, albeit with a reduced diffusion coefficient. Lastly, we measured changes of diffusion upon induction of dispersal and detected an increase in diffusion coefficient inside the biofilm before any loss of biomass. Thus, the change in diffusion is a proxy to detect early stages of dispersal. Our work shows that 3D diffusion maps are very sensitive to physiological changes in biofilms, viz. dispersal. However, this study also shows that diffusion, as mediated by the biofilm matrix, does not account for the high level of antibiotic tolerance associated with biofilms.

SUBMITTER: Sankaran J

PROVIDER: S-EPMC6841743 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

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Single microcolony diffusion analysis in Pseudomonas aeruginosa biofilms.

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