Impact of DNA Polymerase IV on genome evolution in the opportunistic pathogen Pseudomonas aeruginosa

CASTELL SOFÍA; TUMAS IGNACIO; MISERENDINO MARÍA CLARA; PEZZA ROBERTO; CESCHIN DANILO GUILLERMO; MARIELA R. MONTI

Congreso; LVIII Reunión Anual de SAIB; 2022

The human pathogen, Pseudomonas aeruginosa (PA), is a major etiologic agent in a number of acute and chronic 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 augment 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 chronic 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 under oxidative stress. 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 prevent Pol IV mutagenesis. It was also included the Pol IV-deficient strains mutT dinB (TD) and mutT mutSβdinB (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 for 2650 generations through repeated bottlenecks of a single, randomly chosen individual colony, thereby greatly reducing the effectiveness of selection. 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 MA experiment. All MA lines exhibited similar genome mutation rates (4 10-9 per nucleotide) and mutation spectra were dominated by base substitutions characteristic of oxidative DNA damage (AT>CG). However, Tβ MA lines showed increased mutations in particular cellular pathways that are inactivated in acute to chronic switch of PA infection, such us mobility and pigmentation. This mutation preference for these pathways was not observed in the MA lines derived from T, TD and TDβ. Phenotypic analysis of the MA lines showed that mutations introduced by Pol IV effectively inactivated the target genes. A proportion of Tβ MA lines evolved to a hipopigmentation phenotype whereas T, TD and TDβ MA lines showed an increased production of pyocyanin and pyoverdine pigments. Also, flagellar mobility was decreased in some Tβ MA lines, an effect did not show by T, TD and TDβ MA lines. This analysis is currently being extended to other central phenotypes in the PA adaptation. In conclusion, our work reveals that Pol IV activity and its regulation by MutS might have an essential role in the acquisition of inactivating mutations important for the acute-chronic switch.