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

Functional and Structural Analysis of a Highly-Expressed <i>Yersinia pestis</i> Small RNA following Infection of Cultured Macrophages

<div><p>Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in <i>Yersinia pestis</i> that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in <i>Y</i>. <i>pestis</i>. In particular, the sRNA Ysr170 was highly expressed in intracellular <i>Yersinia</i> and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.</p></div

List of small RNAs with mean normalized counts > 20000 in the intracellular fraction.

<p>List of small RNAs with mean normalized counts > 20000 in the intracellular fraction.</p

<i>Yersinia pestis</i> small RNA studies.

<p><i>Yersinia pestis</i> small RNA studies.</p

List of validated sRNAs in this study.

<p>List of validated sRNAs in this study.</p

Analysis of KD Ysr170 strain.

<p>(A) Total RNA was isolated from wild-type (WT) and knock-down (KD) strains of Ysr170 in <i>Y</i>. <i>pestis</i> and <i>Y</i>. <i>pseudotuberculosis</i>, and Ysr170 expression was analyzed by northern blot. (B) Growth curves of <i>Y</i>. <i>pestis</i> (black lines) and <i>Y</i>. <i>pseudotuberculosis</i> (red lines) wild-type (solid) and Ysr170 KD (red) strains were examined in response to different stress conditions up to 8 hrs at 2 hr intervals. Stress conditions include temperature (37°C), mild acidity (pH 5.5), host serum (10% FBS), iron starvation (100μM 2,2’-dipyridyl), and oxidative stress (1mM H₂O₂). A representative experiment from three independent experiments is shown.</p

List of small RNAs with mean normalized counts > 20000 in the intracellular fraction.

<p>List of small RNAs with mean normalized counts > 20000 in the intracellular fraction.</p

Interactions of aminoglycosides with Ysr170 were investigated using SHAPE probing.

<p>(A) 500 nM sRNA was titrated with 1mM of antibiotic. Nucleotides with high SHAPE reactivity are shown in red and nucleotides with low reactivity are shown in black. Helix 1 (H1) contains 4 internal loops/bulges and is capped by a AUU triloop with UA neck base pair. Helix 2 (H2) contains a large internal loop/bulge, a single adenine bulge, and is capped by a 9-nucleotide loop. Helices 3 (H3) and 6 (H4) contain no internal loops and are both capped by 8-member loops (note that helix 6 contains a single AA bulge). Helix 4 (H4) contains a 1–2 bulge and is capped by a tetraloop. Finally, helix 5 (H5) contains a 4–2 internal loop and is capped by a pentaloop. Proposed secondary structure showing regions with reduced SHAPE reactivity in the presence of gentamycin is shown in gold. (B) Comparison of normalized SHAPE reactivity for Ysr170 between reactions in the presence (black) or absence (red) of kanamycin (upper) or gentamycin (lower).</p

Comparison of the log2 fold change from extracellular to intracellular <i>Yersinia</i> and the mean normalized counts in intracellular <i>Yersinia</i> for all 180 small RNAs.

<p>Comparison of the log2 fold change from extracellular to intracellular <i>Yersinia</i> and the mean normalized counts in intracellular <i>Yersinia</i> for all 180 small RNAs.</p