EXPLORING PATHO-ADAPTIVE MUTATIONS PROMOTING INVASIVENESS AND PERSISTENCE OF Pseudomonas aeruginosa WITHIN LUNG EPITHELIAL CELLS

LOPEZ VERONICA ALEJANDRA

Congreso; XVIII Congreso Argentino de Microbiología General.; 2023

Pseudomonas aeruginosa (PA) is a recognized pathogenic bacterium associated withdiverse human infections. Despite its traditional classification as an extracellular pathogen,recent research has unveiled its capacity for epithelial cell invasion and persistence. Thisphenomenon likely contributes to immune evasion and antibiotic resistance, thusunderpinning the chronic nature of PA infections. However, the underlying factors governingthis invasive trait remain enigmatic, and the full spectrum of alternative pathways throughwhich PA can acquire its invasive phenotype remains to be elucidated.In our previous investigations, we thoroughly examined the invasive and persistentbehaviors of both wild-type (wt) and hypermutator PAO1 strains within A549 lung epithelialcells. Through a carefully designed long-term evolutionary experiment, we conducted tensuccessive infection assays. Each assay comprised an antibiotic exclusion protocol,involving controlled lysis of A549 cells and subsequent isolation of intracellular bacteria.These intracellular bacterial populations were afterward employed as the primary inoculumfor following infection assays.In this study, our focus turned to characterizing the evolved bacterial populations.Significantly, we assessed diverse virulence factors and employed Caenorhabditis elegansand Arabidopsis thaliana as infection models. Our investigations revealed a gradualaugmentation in invasive prowess across successive experimentation rounds. Thehypermutator strain notably exhibited marked enhancements in invasive capacity comparedto the wild-type, which retained values akin to the parental strains. Notably, confocalmicroscopy showed the intracellular distribution of evolved PA hypermutator populations,unveiling distinctive bacterial clustering within the cytosol, bypassing the endolysosomalpathway.To elucidate the molecular bases of these observations, we performed whole-genomesequencing on parental strains and isolated 50 clones from the evolved bacterialpopulations (25 wt and 25 hypermutator). Comparative genomics analysis revealed nosequence variations in wt clones respect to parental strains. Conversely, each of the 25hypermutator-evolved clones displayed about 50 distinct SNP mutations, totaling 746variations within coding regions. Focusing on virulence genes, we identified mutationslinked to adherence, antimicrobial activity, antiphagocytosis, enzymatic functions, ironuptake, and secretion systems. Notably, integral T6SS genes tseI and vgrB1 were majormutation targets.Our findings provide insight into the ongoing adaptive progression of PA within eukaryoticcells and underscore how hypermutability amplifies invasiveness while concurrentlydiminishing virulence and cytotoxicity. Further studies are warranted to comprehensivelyunravel the nuanced role of this intricate process in shaping PA invasive phenotype.