DNA POLYMERASE IV, A KEY PLAYER IN THE GENOME EVOLUTION OF THE OPPORTUNISTIC PATHOGEN PSEUDOMONAS AERUGINOSA

FERNANDEZ CONSUELO; CASTELL SOFÍA; PEZZA ROBERTO; MONTI MARIELA R

Congreso; XVIII Reunión Anual de SAMIGE; 2023

he human pathogen, Pseudomonas aeruginosa (PA), is a major etiologic agent in a number of infections. During infection development, PA gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This adaptive process is mainly mediated by inactivating mutations that turn off acute virulence factors (i.e., motility appendages and pigments) and enhance traits associated with chronic infection (i.e., antibiotic resistance). PA undergoes these evolutionary changes in response to selective forces, like the highly oxidative environment, during the infection process. Identification of key players involved in this adaptation process may help to design more effective antimicrobial treatments. In this sense, the mutagenic DNA polymerase (Pol) IV catalyzes the error-prone bypass or incorporation of oxidized nucleotides. We previously reported that the Mismatch Repair protein MutS regulates the access of Pol IV to replication sites in PA by controlling Pol IV interaction with β clamp, which localizes Pol IV to sites of DNA synthesis. In the present work, we evaluated the involvement of Pol IV in the PA genome evolution. With this aim, we analyzed a mutT deficient strain (T), where prevention of nucleotide oxidation is impaired, and a mutT mutSβ (Tβ) strain, where MutS does not avoid Pol IV mutagenesis by incorporation of oxidized nucleotides. It was also included the Pol IV-deficient strains mutT dinB (TD) and mutT mutSβ dinB (TβD). In order to study the role of Pol IV in the mutagenesis of the PA entire genome, we performed mutation accumulation (MA) experiments in which de novo spontaneous mutations accumulate across the genome randomly as selection is expected to be dramatically reduced. MA lines were initiated by creating replicates of each of the four founder strains and propagating lines through repeated bottlenecks of a single, randomly chosen individual colony. The whole genome of the founder strains and each MA line were then analyzed by next generation sequencing to evaluate the mutational events that occurred over the time frame of the experiment. The strains show no statistically significant differences in the mutation rates. The mutation spectra were dominated by base substitutions characteristic of oxidative DNA damage (AT>CG). Notably, Tβ showed a mutation tendency towards certain functional PseudoCAP categories, e.g. in genes related to chemotaxis, motility and antibiotic resistance, which was not observed in lines derived from T, TD and TβD. These results suggest that Pol IV activity and its regulation by MutS might have a role in the acquisition of mutations important for the acute-chronic switch in PA infection. Finally, and in order to analyze the importance of Pol IV in genome evolution in vivo, we are looking for Pol IV fingerprints characterized in our MA experiment in PA genomes from clinical and environmental isolates, available on Pseudomonas.com.