High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of β-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the β-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulatory element mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance killing of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.