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

CASTELL SOFIA; TUMAS IGNACIO NICOLAS; MISERENDINO CLARA; PEZZA ROBERTO; CESCHIN DANILO; MONTI MARIELA ROXANA

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad argentina de investigaciones en bioquímica y biología molecular

The human pathogen, Pseudomonas aeruginosa (PA), is a major etiologic agent in a number of acute and chronicinfections. During infection development, PA gradually shifts from an acute virulent pathogen of early infection to ahost-adapted pathogen of chronic infection. This adaptive process is mainly mediated by inactivating mutations thatturn off acute virulence factors (i.e., motility appendages and pigments) and augment traits associated with chronicinfection (i.e., antibiotic resistance). PA undergoes these evolutionary changes in response to selective forces, likethe highly oxidative environment, during the chronic infection process. Identification of key players involved in thisadaptation process may help to design more effective antimicrobial treatments. In this sense, the mutagenic DNApolymerase (Pol) IV catalyzes the error-prone bypass or incorporation of oxidized nucleotides. We previouslyreported that the Mismatch Repair protein MutS regulates the access of Pol IV to replication sites in PA bycontrolling Pol IV interaction with β clamp, which localizes Pol IV to sites of DNA synthesis. In the present work, weevaluated the involvement of Pol IV in the PA genome evolution under oxidative stress. With this aim, we analyzeda 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. It was also included the Pol IV-deficient strains mutT dinB (TD) andmutT mutSβdinB (TβD). In order to study the role of Pol IV in the mutagenesis of the PA entire genome, weperformed mutation accumulation (MA) experiments in which de novo spontaneous mutations accumulate acrossthe genome randomly as selection is expected to be dramatically reduced. MA lines were initiated by creatingreplicates of each of the four founder strains and propagating lines for 2650 generations through repeatedbottlenecks 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 toevaluate the mutational events that occurred over the time frame of the MA experiment. All MA lines exhibitedsimilar genome mutation rates (4 10-9 per nucleotide) and mutation spectra were dominated by base substitutionscharacteristic of oxidative DNA damage (AT>CG). However, Tβ MA lines showed increased mutations in particularcellular pathways that are inactivated in acute to chronic switch of PA infection, such us motility and pigmentation.The 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 targetgenes. A proportion of Tβ MA lines evolved to a hipopigmentation phenotype whereas T, TD and TDβ MA linesshowed an increased production of pyocyanin and pyoverdine pigments. Also, flagellar motility was decreased insome Tβ MA lines, an effect not shown by T, TD and TDβ MA lines. This analysis is currently being extended to othercentral phenotypes in the PA adaptation. In conclusion, our work reveals that Pol IV activity and its regulation byMutS might have an essential role in the acquisition of inactivating mutations important for the acute-chronicswitch in PA infection.