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.

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro

“…We further investigated tRA99020 dosage effect on the activation of a gene associated with antibiotic tolerance in B. thailandensis persister cells. In our previous study, using comparative transcriptomics and functional genomic assays, we have demonstrated that polyketide synthase (PKS) function played a significant role in B. thailandensis persistence to a variety of antibiotics [ 10 ]. Here, we utilized the pks gene expression as a biomarker of persistence onset.…”

“…We have previously shown that polyketide metabolites produced by B. thailandensis PKS systems act as inhibitors of mammalian histone deacetylase (HDACi) enzymes in in vitro assays [ 10 ]. Since HDAC activity in mammalian cells is essential for rapid immune responses to infections [ 31 , 32 ], we tasked ourselves with testing the efficacy of B .…”

“…The clinical impact of bacterial persistence has stimulated fundamental research on elucidation of the molecular mechanisms crucial for bacterial tolerance to antibiotics [ 10 , 11 ]. A remodeling of the lipid component of the bacterial cell walls and the activation of the transmembrane transport systems for an efflux of toxic metabolites have been identified as phenotypic adaptations that increase antibiotic tolerance in various bacterial species [ 12 , 13 ].…”

“…A remodeling of the lipid component of the bacterial cell walls and the activation of the transmembrane transport systems for an efflux of toxic metabolites have been identified as phenotypic adaptations that increase antibiotic tolerance in various bacterial species [ 12 , 13 ]. Dormant Mycobacterium tuberculosis and persistent Burkholderia thailandensis cells activate genes regulating fatty acid degradation ( fadE and fadD ) [ 10 , 14 ]. Pyrazinamide (PZA) is the only known drug that was found to be effective against dormant M. tuberculosis .…”

“…Both Burkholderia species share common antibiotic resistance mechanisms and an adaptation to host antibacterial responses [ 24 , 25 ]. We have previously identified polyketide synthase (PKS) systems that are significantly upregulated in antibiotic-tolerant populations of B. thailandensis and have demonstrated their role in bacterial persistence to antibiotics [ 10 ]. Furthermore, these PKS systems have been linked to the production of natural products that inhibit mammalian histone deacetylase (HDAC) function [ 26 ].…”