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

Infection efficiency of Ysr170 KD strains.

<p>(A) THP-1 cells were infected with <i>Yersinia</i> wild-type or KD Ysr170 strains at MOI 5 for 30 min to allow for pathogen invasion. Serial dilutions of THP-1 lysates (1:100) were spread on BHI agar plates, and colonies were counted after 48 hrs. The average mean and standard deviation from three representative experiments are shown. The ‘*’ denotes statistical significance (p<0.05) for number of colonies in the Ysr170 KD strains compared to wild-type (WT). (B) THP-1 cells were infected at MOI 5 with <i>Y</i>. <i>pestis</i> and <i>Y</i>. <i>pseudotuberculosis</i> wild-type or KD Ysr170 strains. After 24 hrs, cell supernatants were collected and analyzed by ELISA. The average mean and standard deviation from three representative experiments are shown. The ‘*’ denotes statistical significance (p<0.05) for TNF-α release in the Ysr170 KD strains compared to wild-type (WT). (C) THP-1 cells were infected at MOI 5 with <i>Y</i>. <i>pestis</i> pCD1+ and pCD1- strains that expressed wild-type or KD Ysr170. After 24 hrs, cell supernatants were collected and analyzed by ELISA. The average mean and standard deviation from three representative experiments are shown. The ‘*’ denotes statistical significance (p<0.05) for TNF-α release in the Ysr170 KD <i>Y</i>. <i>pestis</i> pCD1+ strains compared to wild-type (WT). (D) THP-1 cells were infected at MOI 4 with <i>Y</i>. <i>pestis</i> pCD1+ and pCD1- strains that expressed wild-type or KD Ysr170. After 24 hrs, total RNA was extracted and RT-PCR analysis was performed using specific probes to IL-8 and EGR1. The relative RNA levels are presented as fold expression in the KD Ysr170 strains compared to wild-type. For each sample, the RNA levels were normalized to 5S rRNA. The ‘*’ denotes statistical significance (p<0.05) for fold RNA change in IL-8 and EGR1 expression between pCD1+ and pCD1- strains. The average mean and standard deviation from three representative experiments are shown.</p