Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3' end formation in response to azithromycin

Abstract

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3' ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3' end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3' ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3' end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3' end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

Fig 1. 3pMap workflow and data analysis.

(A) 3pMap library synthesis. A 3’ adapter is ligated to total RNA, followed by rRNA depletion. The cDNA is generated by reverse transcription with oligos containing sequences complementary to the adapter (first strand, FS) and to random priming sites (second strand, SS). The cDNA libraries are PCR amplified to generate Illumina sequencing libraries. (B) Assaying changes in full length transcript synthesis. 3’ end reads and peaks were categorized as intragenic (I) and full length (FL) depending on their locations. Differential changes in gene expression were measured using DEseq2 of all 3’ end reads (see Methods). Relative changes in premature 3’ end formation were measured by comparing the I/FL peaks in AZM-treated vs. untreated samples. (C) Predicting RNA regulatory elements in transcript leader neighborhood. 3’ end reads are assigned to genes /operons and categorized as early termini (transcript leader + 40 nucleotides) (ET), intragenic (I), and 3’UTR or full length (FL) reads. Genes with significant 3’ end peaks in the ET zone and %ET >30 are considered outlier candidates.

Fig 2. AZM alters full length transcript synthesis of P. aeruginosa genes.

(A and B) Scatter plot of DEseq2 analysis of expression changes in PA14 planktonic cultures and biofilms treated with AZM. Red dots indicate DEseq changes at 10% FDR. (C) Genes showing >2 fold down (top) or up (bottom) differential regulation upon AZM treatment. AZM induced regulation of planktonic and biofilm genes, indicated in red and blue, respectively, show little overlap. Key PA14 genes regulated by AZM in biofilm (D) cultures and planktonic (E) cultures. Downregulated genes are colored blue, whereas upregulated genes are colored green. Many of these genes have pleiotropic functions, thus impact additional cell processes beyond the primary category in which they are shown here.

Fig 3. AZM alters intragenic 3’ end peak formation in PA14 genes.

(A and B) AZM dependent increase in intragenic premature 3’ end peaks in lasI and rhlR genes. IGV views of lasI and rhlR in control (-AZM) and AZM treated (+AZM) treated samples from planktonic and biofilm cultures are shown. Intragenic peaks whose relative levels are altered by AZM are indicated with blue asterisks. The lasI gene in planktonic cultures and biofilm showed ~16 fold and ~41 fold increase in I/FL peak ratio upon AZM treatment, respectively. AZM treatment resulted in an ~8 fold increase in I/FL peaks for rhlR in biofilms. The peak is not prominent in planktonic cultures. (C) IGV view of the hemN gene downregulated by AZM (DEseq2 log₂fold change = -1.2) shows a 60 fold increase in I/FL peak ratio. The effect is less pronounced in planktonic cells (D) IGV view of lasB downregulated by AZM (DEseq2 change = ~1.95) shows moderate increase in I/FL ratio (1.74 fold) in the presence of AZM. (E) 23 fold upregulation of obgE is accompanied by a 42 fold reduction in transcription attenuation in its transcript leader. (F) A closer view of the transcript leader and CDS start shows termination before the rare codon for Lys at the obgE start codon.

Fig 4. Predicting genes regulated by premature 3’ end formation in transcript leaders.

IGV views show strand-specific 3’ end counts. (A) Example of a gene (dksA PA14_62490) not predicted to be regulated by premature transcription termination in its transcript leader. The major 3’ end peak is in the 3’ UTR. (B) IGV view of early termination in the amiE operon, known to be regulated by transcription attenuation, shows major 3’ end peak in transcript leader. (C) IGV view of lasR shows premature 3’ end peaks in its transcript leader. The sequence of the lasR early termination region and predicted alternate secondary structures in the transcript leader are shown below. The asterisk denotes a premature 3’ end peak. Alternate structures with potential to effect early termination (terminator and transcriptional read-through (anti-terminator was predicted with PASIFIC [68] using the sequence upstream of the asterisk (corresponding to a 3’ end significant peak) as the input. (D) IGV view of DNA gyrase A (gyrA) shows premature 3’ ends in its transcript leader. The sequence of gyrA transcript leader and 40 nt in the CDS and the alternate structures predicted by PASIFIC are shown below, as in C.

Fig 5. Model of P. aeruginosa response to azithromycin.

(A) Altered premature 3’-end formation is observed in up and down-regulated genes/operons of P. aeruginosa in response to AZM. AZM induces changes in premature transcription termination, transcription pausing and/or transcription elongation rate, due to transcription-translation uncoupling or altered RNA polymerase pausing in response to ppGpp, thereby resulting in gene expression changes. Alternatively, nuclease action on premature and full-length transcripts could contribute to gene expression changes in response to AZM. Different molecular mediators of transcriptional control that could affect transcriptional changes are indicated–Rho and Mfd (transcription termination), ppGpp (RNAP pausing), and Hfq (sRNA action and nuclease recruitment). (B) Impact of AZM on gene expression of key P. aeruginosa regulators. Network diagram depicts several genes found to be differentially regulated and exhibit premature transcription termination within the GacS/GacA signal transduction system, quorum sensing systems, and stringent response regulators. These genes, and their regulons, have been shown to be highly interconnected, therefore only a subsection of interactions are shown here. Oval outlines indicate that a gene was differentially expressed upon AZM treatment as detected by DESeq2. Significant hits for altered relative I/FL ratio upon AZM treatment is shown by color of the text. Genes with altered expression or early transcription termination in biofilms are indicated in blue, in planktonic culture in red, and in both conditions in purple. The nature of the expression changes is indicated in Fig 3D.