Investigating the regulatory impact of the small regulatory RNA BecO on essential virulence factors in Pseudomonas aeruginosa

To survive in the harsh environment of the human host, pathogens such as the gram-negative bacterium Pseudomonas aeruginosa (Pa) must utilize a broad array of virulence factors. Aberrant firing of these often metabolically costly pathways can result in immune detection or out competition, thus their production is tightly regulated. Recent techniques have allowed for the expanded study of small RNA (sRNA) mediated regulation in bacteria and it is increasingly clear that these ubiquitous molecules exert important regulatory effects, even on systems whose regulation was previously thought to be well-understood. Recently, work from the Dove lab utilized RNA interaction by ligation and sequencing (RIL-seq) to assess the global RNA-RNA interactions mediated by the RNA chaperone Hfq in Pa. Among this data I found an abundant uncharacterized sRNA, that we refer to as BecO, which appears to be highly conserved in Pa. To begin understanding the pathways impacted by this sRNA, I performed RNA-seq. Excitingly, my preliminary data shows that BecO significantly regulates the expression of hundreds of genes. Among these genes, those encoding for the Type III secretion system (T3SS) and pyochelin synthesis appeared to be strongly regulated. These pathways are both critical for the virulence of Pa, suggesting an important role for BecO during infection. Here I propose to investigate the outsized regulatory role of this sRNA and build a skillset that can be broadly applied to investigating other sRNA regulatory systems. This will be aided by my sponsor, Dr. Simon Dove’s expertise on sRNA regulation in Pa. In Aim 1 I will determine how BecO controls T3SS gene expression. I will investigate whether BecO exerts its regulatory effects by base- pairing with the mRNA transcripts of one or more of the master transcription regulators that govern T3SS gene expression. I will also determine which of the three putative seed regions that I have identified in BecO are involved in mediating T3SS control. In Aim 2 I propose to determine how BecO controls expression of the pyochelin biosynthesis genes. To do this I propose to develop a more sensitive assay based on RIL-seq to identify additional RNA interaction partners for BecO. This technique would have broad utility in the identification of regulatory targets for sRNAs in Pa and other bacteria. Preliminary experiments have shown the growth of the human pathogen Acinetobacter baumannii is altered when co-cultured with Pa in a manner that is influenced by BecO. In Aim 2, I also plan to determine if this altered growth is due to any positive regulatory effects BecO might have on pyochelin production. Together the work proposed will further our understanding of the complex regulation imposed on P. aeruginosa virulence factors and generate new insights into how sRNAs fit into these regulatory circuits. By performing this work in the highly collaborative and intellectually stimulating environment of the Boston Children’s Hospital and Harvard Medical School communities I will have an excellent opportunity to develop the skills needed to become an independent investigator. Project Number: 1F32AI186459-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Brendan O'Hara | Institution: BOSTON CHILDREN'S HOSPITAL, BOSTON, MA | Award Amount: $75,520 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F07A-M (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32AI18645901A1