RplI interacts with 5’ UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5’ untranslated region (5’ UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5’ UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Extraribosomal Functions of Bacterial Ribosomal Proteins—An Update, 2023

“…The abovementioned activities are related to bL9 within the ribosome. Recently, an unforeseen moonlighting activity of bL9 has been revealed [ 225 ]. In P. aeruginosa , bL9 appears to repress translation of the exsA mRNA by binding to its 5′ UTR.…”

The transcriptional regulators of virulence for Pseudomonas aeruginosa: Therapeutic opportunity and preventive potential of its clinical infections

“…The expression of the pelA -G operon is positively regulated by PA0225. 69 The PA0225-deletion mutant produces less biofilm than the wild-type strain PAO1. The intracellular c-di-GMP concentration dynamically regulates P. aeruginosa biofilm formation .…”

“…As the master regulator of the T3SS in P. aeruginosa , the self-feedback TF ExsA is mediated by six positive regulators (Vfr, VqsM, Fis, RcoA1, PsrA, and HigB) and four negative regulators (PtrA, ExsD, MvaT/U, and HigA) at the transcriptional level. ExsA is positively regulated by DeaD 89 but negatively regulated by Hfq, 90 CspC, 91 and RplI 69 at the translational level. In addition, the transcription of T3SS effectors can be directly regulated by a group of TFs.…”