Carbon source-driven downregulation of the sRNA AmiL enhances <i>Pseudomonas aeruginosa</i> virulence during acute lung infection

<i>Pseudomonas aeruginosa</i> is a major opportunistic pathogen that causes severe acute lung infection. The ability of <i>P. aeruginosa</i> to precisely self-regulate its virulence factors is crucial for enhancing pathogenicity and establishing infection. However, how the bacterium dynamically adjusts virulence gene expression in response to host interaction remains to be elucidated. Here, transcriptome profiling revealed that infection of A549 alveolar epithelial cells significantly upregulated virulence genes associated with the type III secretion system (T3SS) and quorum sensing (QS) in <i>P. aeruginosa</i> PAO1, compared to a standard laboratory growth condition. In contrast, genes of the <i>amiEBCRS</i> operon were markedly downregulated during infection. Notably, AmiL, the leader small RNA (sRNA) of the operon, exhibited consistently reduced expression across multiple <i>P. aeruginosa</i> infection models, including A549 cells, mouse lungs, and clinical isolates. AmiL expression was primarily repressed by succinate-mediated carbon catabolite repression (CCR) during the early stage of infection, and later by the transcription factor ArgR as succinate levels declined. Functional analyses showed AmiL bound to the mRNAs of the T3SS effector gene <i>exoT</i> and the hydrogen cyanide synthesis gene <i>hcnC</i>, repressing their expression and thereby attenuating PAO1 virulence. Furthermore, deletion of <i>amiL</i> gene enhanced the cytotoxicity of PAO1 in A549 cells, <i>Galleria mellonella</i> larvae, and a mouse lung infection model, resulting in more severe tissue damage and increased mortality. Therefore, our findings demonstrate that <i>P. aeruginosa</i> strategically downregulates AmiL in response to carbon source changes to enhance its virulence and pathogenicity during acute lung infection, and highlight a potential target for antibacterial therapy.