A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis

Abstract

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP-binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence.IMPORTANCE The discovery of antibiotics such as penicillin and streptomycin marked a historic milestone in the 1940s and heralded a new era of antimicrobial therapy as the modern standard for medical treatment. Yet, even in those early days of discovery, it was noted that a small subset of cells (∼1 in 10⁵) survived antibiotic treatment and continued to persist, leading to recurrence of chronic infection. These persisters are phenotypic variants that have modified their physiology to survive environmental stress. In this study, we have performed three transcriptomic screens to identify persistence genes that are common between three different stressor conditions. In particular, we identified genes that function in the synthesis of secondary metabolites, small molecules, and complex lipids, which are likely required to maintain the persistence state. Targeting universal persistence genes can lead to the development of clinically relevant antipersistence therapeutics for infectious disease management.

Keywords: Burkholderia; antibiotic resistance; bacterial persistence; stress; transcriptomics.

FIG 1

Characterization of persister phenotype. (A) Analysis of proton motive force (PMF) using fluorescence-activated cell sorting (FACS). Mid-log-growth-phase B. thailandensis cultures (OD₆₀₀ of 0.2 to 0.3) were incubated in LB-Miller broth containing 50 μg/ml SHX, 20 μg/ml Ofl, or 20 μg/ml Mpm for 2 h at 37°C prior to addition of 1 μg/ml DiBAC₄(3). Stationary cells (STAT) were collected from 14-day-old cultures on LB-Miller nutrient agar plates and incubated for 30 min at 37°C in LB-Miller broth containing 1 μg/ml DiBAC₄(3). Flow cytometry analysis was performed on the BD FACSAria instrument using a fluorescein isothiocyanate (FITC) filter (Em: 530/30) after excitation with the 488-nm laser. Mid-log-phase cells not treated with DiBAC₄(3) were used to position the signal threshold gate. One representative experiment out of five independent replicates is shown. (B) Metabolic index of B. thailandensis sorted for DiBAC₄(3) retention. Mid-log-phase B. thailandensis cultures were treated with 1 μg/ml DiBAC₄(3) and sorted into subpopulations with low FI (FITC-A, 500 to ≤1,000 fluorescence units [FU]) and high FI (FITC-A, 10³ to ≤10⁴ FU). Ten thousand cells were collected by FACS and combined with BacTiter-Glo reagent to quantify cellular ATP. Presented are the average and standard deviation values from three independent experiments. (C) Evaluation of bacterial tolerance to cytotoxic antibiotic concentrations. (Left) Mid-log-phase B. thailandensis cultures were treated with 1 μg/ml DiBAC₄(3) and sorted to subpopulations with low FI (FITC-A, 500 to ≤1,000 FU) and high FI (FITC-A, 10³ to ≤10⁴ FU). Ten thousand cells were collected by FACS and grown to early log phase (OD₆₀₀ of ∼0.2) prior to addition of 10 μg/ml Mpm. Persister populations surviving 24 h of antibiotic treatment were calculated as CFU/ml recovered on LB-Miller agar plates after antibiotic treatment relative to the CFU/ml prior to antibiotic exposure. Average and standard deviation values were derived from three independent experiments. (Middle) Mid-log-phase B. thailandensis cultures were subjected to 10 μg/ml Ofl or 10 μg/ml Mpm for 24 h. Percentage of surviving persisters was determined after 24 h of antibiotic exposure. Average and standard deviation values were derived from five independent experiments. (Right) A single CFU from 1-, 7-, 14-, and 21-day-old B. thailandensis cultures was inoculated into LB-Miller broth and cultured at 37°C until OD₆₀₀ reached ∼0.2, followed by addition of 10 μg/ml Ofl for 24 h. Presented are the average and standard deviation values of percent bacterial survival calculated from 10 CFU inocula originating from three independent bacterial populations.

FIG 2

Comparative analysis of genome-wide transcriptomics data from three persistence phenotypic screens. (A) Principal-component analysis of RNA-seq data was performed using DESeq2 as part of PiReT analyses on the EDGE bioinformatics platform. The control and persister populations for the three experimental methods are indicated in the legend. (B) Comparison of the number of differentially expressed genes (log₂-fold) in persister populations enriched via three methods (low PMF, Mpm, and aged). The P value cutoffs applied for differentially regulated genes were ≤0.001 for the populations with low PMF, and P values of ≤0.01 were used for Mpm-treated and aged populations featuring higher variability. Colored ovals in the legend show five functional groups of interest with gene representatives differentially expressed in persister-enriched populations.

FIG 3

Functional pathways differentially regulated in persisters enriched via different experimental methods. Heatmaps show the fold change (FC) in log₂ scale of genes in selected pathways between persisters and control groups. Gene names are labeled using locus tags. FC values were applied in the hierarchical clustering of genes (rows) and experimental conditions (columns).

FIG 4

PKS gene activity is a determinant of B. thailandensis persistence. (A) PKS BTH_I2366 loss of function increased B. thailandensis sensitivity to antibiotics that target DNA replication. Mid-log-growth bacterial cultures (OD₆₀₀ of ∼0.2 to 0.3) were treated with 1× MBC of Mpm (10 μg/ml), Ofl (10 μg/ml), and Cipro (5 μg/ml) for 24 h. The percentages of persister populations were determined as the ratio of antibiotic-surviving CFU/ml versus the cell number (CFU/ml) prior to antibiotic exposure. Presented are data from four independent antibiotic treatment experiments. The significance of the registered sensitivity to antibiotic treatment was evaluated using one-way ANOVA, where # indicates a P value of <0.002. (B) The PKS-encoding BTH_I2366 gene is responsible for membrane depolarization upon Mpm treatment. Wild-type B. thailandensis and mutant strains were grown to mid-log phase (OD₆₀₀ of 0.2 to 0.3) and were treated with 10 μg/ml Mpm for 3 h. Bacterial cultures were incubated in 1 μg/ml DiBAC₄(3) for 30 min prior to flow cytometry analysis on the BD FACSAria using an FITC filter (Em: 530/30). Gate P2 (top panel) represents the population of cells with low PMF that retain the fluorescent dye, shown in green. The total analyzed bacterial population (P1) is shown in red. Data are representative of five independent experiments.

FIG 5

The PKS operon encodes secondary metabolites with HDAC-inhibitory properties that regulate DNA synthesis in B. thailandensis. (A) Treatment with Mpm stimulated accumulation of HDAC inhibitors in wild-type B. thailandensis cultures. Mpm at 10 μg/ml was added to cultures of wild-type B. thailandensis E264 and BTH_I2366 mutants grown to an OD₆₀₀ of ∼0.2. Crude conditioned medium from overnight (18-h) cultures grown without agitation in a 96-well plate was analyzed for inhibition of HDAC activity. Trichostatin A (TSA; 1 μM) was used as an inhibitor control. The percent inhibition of HDAC activity (%) was calculated relative to samples with no drug or conditioned medium. Shown are the average and standard deviation values from three independent experiments. (B) Loss of PKS gene function results in accumulation of DNA in stationary-phase B. thailandensis cultures. Flow cytometry analysis was performed on mid-log-phase (LOG) and stationary-phase (STAT) cultures to determine the DNA content in wild-type B. thailandensis and BTH_I2366 mutant cells. The membrane-permeant Vybrant DyeCycle green stain was added to bacterial cultures 30 min prior to analysis on a BD FACSAria instrument. DNA content was measured as mean fluorescence intensity (MFI) units. Shown are data from 10,000 cells analyzed in one representative experiment out of three independent replicates.

FIG 6

Acetyl-CoA carboxylase inhibits persister formation in Gram-negative bacteria. (A) Combination of Mpm with acetyl-CoA carboxylase inhibitor CP-640186 reduced persisters in B. thailandensis. Mid-log-phase cultures (OD₆₀₀ of 0.2 to 0.3) of B. thailandensis were treated with 10 μg/ml Mpm, 10 μg/ml Ofl, or a combination of antibiotic and 50 μg/ml CP-640186 for 24 h. The percentage of persisters surviving antibiotic treatment was calculated as previously described. Shown are average and standard deviation values derived from four independent experiments. (B) CP-640186 potentiated Mpm activity against persisters in three Gram-negative bacterial pathogens. Mpm at 2× MBC alone or in combination with 50 μg/ml CP-640186 was added to each bacterial species (OD₆₀₀ of ∼0.2). Persister populations were evaluated after 24 h of antibiotic exposure as described above. Shown are average and standard deviation values derived from four independent experiments.