Quantitative mapping of mRNA 3’ ends inPseudomonas aeruginosareveals a pervasive role for premature 3’ end formation in response to azithromycin

Abstract

P. aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including cystic fibrosis patients. 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 QS. The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using an improved genomewide approach to quantitate RNA 3'-ends (3pMap). We also identified hundreds of P.aeruginosa genes subject to premature transcription termination 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 or pausing. Reciprocally, AZM reduced premature transcription termination 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 premature transcription termination downregulates expression of central transcriptional regulators, which in turn both impairs QS and biofilm formation, and stress responses, while upregulating genes associated with persistence.

Neutrophil-mediated delivery of the combination of colistin and azithromycin for the treatment of bacterial infection

“…The quorum sensing regulatory gene rhlR is more vulnerable to the translational repression by azithromycin owing to its rare codon ( Gödeke et al., 2013 ), thus leading to the repression of quorum sensing regulated virulence factors and subsequent inhibition of biofilm formation or disruption of biofilm. A recent study revealed that azithromycin induces changes in the 3′ end formation of mRNAs in P. aeruginosa , which results in repression of genes related to quorum sensing, biofilm formation, and stress responses ( Konikkat et al., 2021 ).…”

Polygenic adaptation, clonal interference, and the evolution of mutators in experimentalPseudomonas aeruginosapopulations

“…Further, gacS has been shown to be involved in the switch from a hyperadherent small colony phenotype back to a wild type phenotype indicating that loss of GacS function is beneficial in biofilms (Davies et al 2007). The additional two biofilm specific loci, PA14_13150 and PA14_71750, are less understood transcriptional regulators, however PA14_71750 has been previously associated with biofilm adaptation in Pseudomonas (Konikkat et al 2020).…”

The structure–function analysis of Obg‐like GTPase proteins along the evolutionary tree from bacteria to humans

“…This conserved GTPase is known to be associated with persister formation in different bacteria, therefore directly or indirectly plays a role in antibiotic resistance. In clinically important organism, Pseudomonas aeruginosa (associated with cystic fibrosis) the expression of full length Obg transcript is enhanced significantly whereas different other genes including those are involved in exerting virulence property has been prematurely terminated in presence of macrolide azithromycin (Konikkat et al, 2021). As Obg has structural interaction with alarmone ppGpp that controls the gene expression under stringent condition by relieving the pause of RNA polymerase across the length of gene, helps in the up‐regulation of obg transcript.…”