Unraveling the molecular dynamics of <i>Pseudomonas aeruginosa</i> biofilms at the air–liquid interface

<b>Aim:</b> The aim of this study was to probe the dynamics of <i>Pseudomonas aeruginosa</i> PA14 air–liquid interface (ALI) biofilms over time through global proteomic analysis. <b>Materials &amp; methods:</b> <i>P. aeruginosa</i> PA14 ALI biofilm samples, collected over 48–144 h, underwent differential expression analysis to identify varying proteins at each time point. <b>Results:</b> A consistent set of 778 proteins was identified, with variable expression over time. Upregulated proteins were mainly linked to ‘amino acid transport and metabolism’. Biofilm-related pathways, including cAMP/Vfr and QS, underwent significant changes. Flagella were more influential than pili, especially in early biofilm development. Proteins associated with virulence, transporters and iron showed differential expression throughout. <b>Conclusion:</b> The findings enhance our understanding of ALI biofilm development. This study looks at how the bacteria <i>Pseudomonas aeruginosa</i> forms a community called a biofilm at the air–liquid interface (ALI), an important environment for bacterial growth. Biofilms at the ALI are resistant to external forces and contribute to antibiotic resistance. Over 48–144 h, we observed a noticeable increase in biofilm thickness. Our data suggested that the flagella, a sort of propeller of the bacterium, plays a crucial role, especially in the initial stages of ALI biofilm formation. Proteins associated with virulence, transporters and iron also showed their significance in ALI biofilms. These findings offer valuable insights into the protein changes and functions involved in <i>P. aeruginosa</i> ALI biofilms, improving our understanding of biofilm development. The most significant increase in <i>P. aeruginosa</i> PA14 ALI biofilm thickness occurred between 48 and 72 h, with continued growth over time. Distinct protein clusters between ALI biofilms and planktonic cells identified through heatmap analysis. Venn diagram revealed 778 commonly shared proteins across all ALI biofilm time points. ‘Metabolism’ was the most abundant category among upregulated proteins in COG analysis. KEGG pathway analysis highlighted ‘amino acid metabolism’ and ‘carbohydrate metabolism’ enrichment in both upregulated and downregulated sets. Modulation of 81 proteins involved in biofilm formation, with significant changes observed from 72 to 144 h. Key regulators such as Vfr-cAMP signaling, quorum sensing and c-di-GMP signaling exhibited significant proteomic modulation. Proteins involved in flagella structure, type I and VI secretion systems and alginate/siderophore synthesis exhibited significant upregulation. Significant changes in 175 transporter proteins, with 89 upregulated proteins at 48 h, mainly belonging to the ABC superfamily. Elevated iron-related protein expression observed at the 48 h point suggested a critical role in early-stage biofilm development. The study provides valuable insights into the proteome dynamics and functional pathways associated with <i>P. aeruginosa</i> PA14 ALI biofilms, enhancing our understanding of biofilm development.