Exploring aggregation genes in aP. aeruginosachronic infection model

Abstract

AbstractBacterial aggregates are observed in both natural and artificial environments. In the context of disease, aggregates have been isolated from both chronic and acute infections.Pseudomonas aeruginosa(Pa) aggregates contribute significantly to chronic infections, particularly in the lungs of people with cystic fibrosis (CF). Unlike the large biofilm structures observedin vitro, Pain CF sputum forms smaller aggregates (∼10-1000 cells), and the mechanisms behind their formation remain underexplored. This study aims to identify genes essential and unique toPaaggregate formation in a synthetic CF sputum media (SCFM2). We culturedPastrain PAO1 in SCFM2 and LB, both with and without mucin, and used RNA sequencing (RNA-seq) to identify differentially expressed genes. The presence of mucin revealed hundreds of significantly differentially expressed (DE) genes, predominantly downregulated, with 40% encoding hypothetical proteins unique to aggregates. Using high-resolution microscopy, we assessed the ability of mutants to form aggregates and identified 13 that were unable to form WT aggregates. Notably, no mutant exhibited a completely planktonic phenotype. Instead, we identified multiple spatial phenotypes described as ‘normal,’ ‘entropic,’ or ‘impaired.’ Entropic mutants displayed tightly packed, raft-like structures, while impaired mutants had loosely packed cells. Predictive modeling linked the prioritized genes to metabolic shifts, iron acquisition, surface modification, and quorum sensing. Co-culture experiments with wild-type PAO1 revealed further spatial heterogeneity and the ability to ‘rescue’ some mutant phenotypes, suggesting cooperative interactions during growth. This study enhances our understanding ofPaaggregate biology, specifically the genes and pathways unique to aggregation in CF-like environments. Importantly, it provides insights for developing therapeutic strategies targeting aggregate-specific pathways.ImportanceThis study identifies genes essential for the formation ofPseudomonas aeruginosa(Pa) aggregates in cystic fibrosis (CF) sputum, filling a critical gap in understanding their specific biology. Using a synthetic CF sputum model (SCFM2) and RNA sequencing, 13 key genes were identified, whose disruption led to distinct spatial phenotypes observed through high-resolution microscopy. The addition of wild-type cells either rescued the mutant phenotype or increased spatial heterogeneity, suggesting cooperative interactions are involved in aggregate formation. This research advances our knowledge ofPaaggregate biology, particularly the unique genes and pathways involved in CF-like environments, offering valuable insights for developing targeted therapeutic strategies against aggregate-specific pathways.