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.

A gene network-driven approach to infer novel pathogenicity-associated genes: application to Pseudomonas aeruginosa PAO1

“…We also found enrichment of KEGG pathways related to quorum sensing and phenazine biosynthesis in module 7, which were both downregulated. As azithromycin dysregulates pili-mediated chemotaxis ( 95 ), two-component system ( 96 ), biofilm formation ( 97 ), phenazine biosynthesis ( 98 ), quorum sensing, and alginate biosynthesis ( 99 , 100 ), enrichment of processes associated with these modules supports our model’s effectiveness in unraveling processes that are relevant during the treatment of PAO1 with azithromycin.…”

Refining the transcriptional landscapes for distinct clades of virulent phages infecting Pseudomonas aeruginosa

“…Furthermore, we find that individual phage TUs are highly interconnected, suggesting that alternative TU usage might be a widespread regulatory strategy in Pseudomonas phages to balance and finetune gene expression levels in their densely coded genomes in response to different stimuli (Lee et al 2019 ; Putzeys et al 2022 , 2023a ). In bacteria, 5′UTR conditional premature transcription is an important regulatory mechanism to modulate gene expression levels under different environmental stressors and conditions (Merino and Yanofsky 2005 , Adams et al 2021 , Konikkat et al 2021 ). ONT-cappable-seq data of the different phages also revealed numerous 5′UTR premature transcription termination events, pointing to 29 potential phage-encoded 5′UTR-derived ncRNA candidates, two of which were previously detected by phiKZ dRNA-seq and grad-seq experiments (Gerovac et al 2021 , Wicke et al 2021 ).…”

Refining the transcriptional landscapes for distinct clades of virulent phages infectingpseudomonas aeruginosa

“…In bacteria, 5’UTR conditional premature transcription is an important regulatory mechanism to modulate gene expression levels under different environmental stressors and conditions (Merino and Yanofsky 2005; Adams et al . 2021; Konikkat et al . 2021).…”