OprD mutant Pseudomonas aeruginosa (PA) is a strain that is resistant to carbapenems by loss of functional OprD porin and that shows increased pathogenicity towards the airway epithelium.

To understand what could lead to the virulence rise and identify new potential therapeutic targets, we sought to determine essential genes (EGs) of OprD mutant PA14 by TnSeq (Transposon Sequencing).

Transposon mutant libraries were constructed for PA14 WT and PA14?oprD, inactivating in each bacterium a different gene from the entire chromosome. These libraries were then cultured in LB medium and bacterial DNA was extracted and sequenced at high throughput in single-read on Nextseq 500. EGs were determined by comparing different methods and analysis parameters with TRANSIT and FiTnEss software. The identified genes were annotated and the results for the two strains were compared. Specific genes of the OprD mutant strain were biologically verified by clean deletion and inducible transcriptional repression with CRISPRi/dCas9.

A list of 510 GEs was determined for PA14?oprD versus 609 GEs for PA14 WT, 86 genes were specific to the WT strain and 31 to the OprD mutant strain. After having annotated these 31 GEs, the focus was made on 2 involved in Krebs cycle precursors obtainment. One of these two genes showed a growth defect during CRISPRi/dCas9 induction in growth curves. The repression of this gene led to a 65% diminution of CFU compared to the control (p<0.05).

Among the EGs found in the mutant strain linked to the loss of the OprD porin, one was confirmed to reduce growth in the mutant strain only, making it a new potential therapeutic target in the case of carbapenem-resistant infection.