Identification of PilD mutants reveals the iterative social evolution in <i>Pseudomonas aeruginosa</i>

Bacterial pathogenicity is influenced by the genetic mutations that arise within the population, among which quorum-sensing (QS) is one of the key targets. Recent studies have reported that mutations in the global transcriptional regulator gene <i>mexT</i> can revert QS activity in QS-inactive LasR mutants, thereby transforming the LasR cheater mutants deficient in public goods production into cooperators. However, the subsequent evolutionary trajectory of this population remains poorly understood. To gain a deeper understanding of the evolutionary trajectory of <i>Pseudomonas aeruginosa</i>, we conducted an <i>in vitro</i> evolution experiment with the LasR-MexT-deficient mutant strain and examined the emerging variant subpopulations. Under the condition of QS activation, we identified a variant subpopulation carrying mutations in <i>pilD</i>, which encodes a prepilin peptidase involved in both type II secretion system (T2SS) and type IV pili (T4P). In contrast to the parental cooperative LasR-MexT mutant, the PilD mutant displays a social cheater phenotype, characterized by a defect in extracellular protease secretion. The inactivation of PilD resulted in reduced QS activity and the production of QS-controlled products. Furthermore, the PilD mutant caused significantly reduced cell cytotoxicity in mammalian cells. Our findings highlight an iterative evolution of social behavior between cooperators and cheaters. In conclusion, our study provides a roadmap of <i>P. aeruginosa</i>'s evolutionary trajectory in the QS activation environment, shedding light on our understanding of the evolutionary dynamics underlying bacterial pathogenicity.IMPORTANCE<i>P. aeruginosa</i> is a leading cause of opportunistic acute and chronic infections in humans, in which its pathogenicity is intricately intertwined with its evolutionary trajectory and the emergence of genetic mutants within the population. Our studies reveal an iterative social development between cooperative and cheating behaviors, providing valuable insights into the intricate dynamics of social interactions within bacterial populations. Furthermore, our investigations demonstrate dynamic mutant pathogenicity changes during the evolutionary process, suggesting that developing strategies to combat antibiotic resistance and pathogenicity in clinical settings.