Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

Abstract

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.

Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells

“…As suggested above, it is important to recognize that neither VBNC cells nor persisters are metabolically inept. Although there is a large decline in metabolic activity in these cells, they continue to energize their membranes and produce a more specialized set of proteins (10,79). There are several mechanisms by which persister cells are thought to form, and there is growing evidence that these same mechanisms are involved in directing the VBNC state.…”

Characterization of the Pseudomonas aeruginosa NQR complex, a bacterial proton pump with roles in autopoisoning resistance

“…Indeed, P. aeruginosa strains isolated from cystic fibrosis patients show an increase in NQR expression (84,85). Moreover, Pa-NQR subunits have been shown to be expressed by P. aeruginosa during biofilm formation (86,87), especially in the oxygen-rich, metabolically active regions of the biofilm, where bacteria are exposed to competing bacteria, antibacterial metabolites and antibiotics, and other varying environmental factors.…”

Genetic and Transcriptomic Analyses of Ciprofloxacin-Tolerant Staphylococcus aureus Isolated by the Replica Plating Tolerance Isolation System (REPTIS)

“…One reason for this is that, by definition, tolerant cells survive but do not grow in the presence of antibiotics (23, 36, 37), making it difficult to select tolerant bacteria. While many attempts have been made to develop methods to select tolerant cells in a heterogeneous population (11,(19)(20)(21), none of these can be used to routinely quantify the tolerance of a bacterial isolate in the clinic. Very recently, a method, called TDtest, which can easily detect antibiotic tolerance using a modified disk diffusion assay was described.…”

“…Such a method will subsequently enable the design of drugs to eradicate persistent infections. Several attempts have been made to solve the mysteries of antibiotic tolerance, particularly by isolating and quantifying tolerant variants from a heterogeneous population, yet none have been simple or cost-effective enough for use in clinics on a routine basis (11,(19)(20)(21).…”