Pseudomonas aeruginosa is an opportunistic pathogen associated with severe nosocomial infections. Its virulence and adaptation to environmental conditions are coordinated by complex signaling networks. While small RNAs (sRNAs) are established as post-transcriptional regulators of bacterial gene expression, the complete repertoire and functional roles of these non-coding molecules in P. aeruginosa have remained partially defined.

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Source: CDC/ Dr. Lucille K. George

This is a photomicrograph of a Brown and Brenn stained tissue specimen extracted from a patient with a case of botryomycosis, due to the Gram-negative, rod-shaped bacterium Pseudomonas aeruginosa.

In a recent study published in Genes & Diseases, researchers from City University of Hong Kong, The Hong Kong University of Science and Technology, Guangdong Academy of Agricultural Sciences, Sun Yat-Sen University and Virginia Commonwealth University utilized a computational-experimental approach to map the sRNA landscape of this pathogen.

To detect conditionally expressed transcripts, the investigators developed a combined computational-experimental prediction pipeline to analyze intergenic RNA signals. This analysis established a pan-sRNome database encompassing 1,663 annotated sRNA sequences across 484 P. aeruginosa strains, identifying 58 representative sRNA homologs. Of these, nine novel intergenic sRNAs were validated via Northern blotting. Evolutionary analysis classified three of these newly identified transcripts (PA0806.1, PA3471.1, and PA4642.1) as core sRNAs present in all evaluated strains, while the remaining six (PA0982.1, PA1014.1, PA1270.1, PA1367.1, PA2734.1, and PA2736.2) were categorized as accessory elements.

Modulatory effects

Functional characterization of these novel regulators revealed distinct modulatory effects on pathogen virulence. The accessory transcript PA0982.1 was identified as a positive regulator of the rhl quorum sensing system. Biophysical assays demonstrated that PA0982.1 directly binds to the 5’ untranslated region of the rhlI mRNA, facilitating translation initiation. Genetic deletion of PA0982.1 reduced synthesis of the C4-HSL autoinducer, downregulated the downstream regulator rhlR, and decreased pyocyanin production.

Additionally, overexpression of PA1014.1, PA2734.1, and PA2736.2 increased pyocyanin levels through the upregulation of phenazine biosynthesis genes. Strains overexpressing PA1014.1 and PA2734.1 exhibited increased biofilm formation accompanied by elevated expression of the matrix-associated genes cupE and pslC. Conversely, PA2734.1 and PA2736.2 restricted swarming motility by downregulating the fimbrial biogenesis gene pilE.

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(A) Pan-sRNome profiling of P. aeruginosa. Classification of 58 annotated sRNAs from 484 P. aeruginosa genome data into 3 categories: core sRNAs (sRNAs found in all 484 strains), accessory sRNAs (sRNAs found in 2 to 483 strains), and unique sRNAs (sRNAs only found in 1 strain). Eight representative strains, including laboratory, clinical, and reference isolates, are highlighted, with nucleotide sequence identity indicated in the right-hand legend. (B) Distribution of the 58 annotated sRNAs within the P. aeruginosa Pan-sRNome, categorized by prevalence across strains. (C) Categorization of 9 new sRNAs within the Pan-sRNome of P. aeruginosa. (D) sRNA composition across 8 representative P. aeruginosa strains. A core set of 38 sRNAs was universally detected, while a unique sRNA was identified exclusively in the clinical strain PABL017. The number of accessory sRNAs varies from 6 to 15 across strains. (E) Genomic localization features of 58 sRNAs in P. aeruginosa. The pie chart shows the genomic positions of 58 representative sRNA sequence homologs, with 9 located within coding sequences (CDSs), 37 in intergenic regions (IGRs), and 12 spanning both CDSs and IGRs. Credit: Tianmin Li, Beifang Lu, Fang Chen, Letong Xu, Yizhou Zhang, Fangzhou Xie, Yue Sun, Chunyan Yao, Jiadai Huang, Tianlong He, Youyue Li, Yiqing Ding, Haiyan Hu, Shaojun Tang, Xin Deng

The study also demonstrated that overexpressing PA1270.1, PA1367.1, and PA3471.1 downregulates Type VI secretion system components, including hcpB and hsiB3, reducing competitive fitness against E. coli. Furthermore, evolutionary analysis across 23 distinct bacterial and archaeal species demonstrated selective conservation of these transcripts, illustrating their integration into bacterial genome structures.

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In conclusion, these findings demonstrate that core and accessory sRNAs coordinate post-transcriptional networks in P. aeruginosa. This pan-sRNome framework provides a metabolic and evolutionary model for investigating sRNA-mediated regulation, offering potential targets for future antimicrobial strategies.

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