ThepanC-encoded pantothenate synthetase to tackle carbapenem-resistant OprDPseudomonas aeruginosamutant revealed through Tn-Seq

AbstractFor the World Health Organization, carbapenem-resistantPseudomonas aeruginosa is a critical priority for which new antimicrobial drugs are needed. Consequently, understanding the underlying mechanisms of resistant bacteria infection will enable the identification of new therapeutic targets. Loss of the OprD porin is the main determinant of resistance to the last resort carbapenem antibiotics and has been described to enhance fitnessin vivoand virulence. Transposon sequencing is a high-throughput sequencing technique that makes it possible to identify essential genes (EGs) that may turn out to be therapeutic targets. However, such a strategy has not yet been used for OprD-deficientP. aeruginosa. In this study, we identified the EGs specific to PA14 OprD mutant for LB growth and we established a list of 30 EGs among these, we highlighted thepanCgene encoding pantothenate synthetase as a promising target. Using CRISPRi, we confirmed that silencingpanCreduced LB growth, and decreasedsigXexpression, whose overexpression is associated with membrane fluidity, as well as the expression of genes involved in the fatty acid synthesis (FAS). Taking into account the weakness of PA14 OprD mutant due to an altered membrane consecutive to a decrease in unsaturated FAS in the absence ofpanC,we showed that silencingpanCextended the destruction time of 16HBE airway cells. Overall, our findings highlighted the anti-virulence potential ofpanCinhibition and shed new light on its inhibition as a target for treating carbapenem-resistant OprD-defective PA lung infections.