Lien vers Pubmed [PMID] – 40823844
Lien DOI – 10.1128/mbio.01343-25
mBio 2025 Aug; (): e0134325
The Enterobacterales outer membrane is impermeable to many antibiotics, but they can enter the cell via channel proteins, called porins. In Klebsiella pneumoniae, the porin OmpK36 is a major determinant of antibiotic resistance. Insertion of amino acids into the L3 constriction loop is frequently reported, leading to decreased channel size and reduced antibiotic susceptibility. While the involvement of the L3 loop in resistance is well-established, studies focused on a limited number of sequences, without considering any role for diversity outside L3. To fill this gap, we carried out a large-scale genomic comparison combined with experimental analysis. We analyzed 16,086 K. pneumoniae genomes to decipher the diversity of OmpK36 at the species level. We identified 385 protein variants classified into 7 main backbones, some associated with high-risk clones and, consequently, with major resistance classes. Comparison of predicted three-dimensional structures indicates variation in pore size depending on backbones. We constructed mutants carrying the main variants and compared their fitness and susceptibility to antibiotics and phages. Despite OmpK36 diversity and predicted pore radius differences, variations beyond the constriction loop do not seem to significantly impact antibiotic susceptibility and fitness. However, this diversity may have been partly shaped by phages targeting OmpK36, as evidenced by varying susceptibility to a T4-like phage depending on the backbones and the rapid acquisition of mutations in ompK36 among initially susceptible mutants following phage exposure. These findings suggest that while antibiotic pressure selects for L3 loop variations, phage predation may be a key driver of broader OmpK36 diversity.Porins are outer membrane proteins involved in the passive diffusion into the periplasm of substrates such as nutrients. They are also a major route for antibiotics. Mutations in the main porins of K. pneumoniae, OmpK35 and OmpK36, have been described and mainly lead to disruption of the former and a pore size reduction of the latter through amino acid insertion in the constriction loop. ompK36 mutations are clinically significant, with a reduction in susceptibility to β-lactams, particularly when strains also produce a carbapenemase. However, little is known about the overall diversity of OmpK36. Through large-scale genome analysis and experimental work, we sought to decipher the selective pressures that shape the diversity of this protein. Our results suggest that antibiotics are not the main driver of OmpK36 diversity at the level of the entire protein sequence and that phages can instead rapidly select variations in the extracellular loops.