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

Abstract

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 likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator 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 lysis 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.

Clonal transmission of polymyxin B-resistant hypervirulent Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaKPC-2 in a tertiary hospital in China

“…The acquisition of drug resistance occurs at the cost of reducing capsular polysaccharides and virulence. Murtha et al [ 33 ] studied Enterobacterales and found that meropenem could induce PhoPQ and increase modifications of L-Ara4N. Exposure to different antibiotics can lead to changes in bacterial tolerance.…”

Peptidoglycan Recycling Promotes Outer Membrane Integrity and Carbapenem Tolerance in Acinetobacter baumannii

“…Previous work showed that Vibrio cholerae ( 17 , 18 ), Pseudomonas aeruginosa ( 19 ), and pathogens of the Enterobacterales order ( 21 , 22 ) form viable, nondividing spheroplasts when exposed to lethal concentrations of β-lactam antibiotics over several hours. Importantly, spheroplasts revert to normal rod-shaped growth when the antibiotic concentration is sufficiently reduced ( 21 ), demonstrating a short-term survival mechanism that directly contributes to antibiotic treatment failure.…”

“…While we showed that several tolerance factors are transcriptionally regulated, we do not know which transcriptional regulators control these responses. A previous study found that PhoPQ-dependent outer membrane modifications promoted survival in cell wall-deficient spheroplasts ( 22 ), presumably by fortifying the outer membrane to counter large loads of turgor pressure typically absorbed by the cell wall. Specifically, PhoPQ was activated in response to meropenem treatment.…”

Cell wall deficiency – an alternate bacterial lifestyle?

“…baumannii upregulated genes that fortified the OM and induced autolysins that may act to remodel the cell envelope [110]. In Enterobacter cloacae , the two-component system PhoPQ was found to be necessary for survival of meropenem-induced CWD cells over 24 h [111]. Further analysis of genes controlled by the PhoPQ regulon identified arnT , which encodes a transferase that adds aminoarabinose to lipid A in the OM [111].…”

“…In Enterobacter cloacae , the two-component system PhoPQ was found to be necessary for survival of meropenem-induced CWD cells over 24 h [111]. Further analysis of genes controlled by the PhoPQ regulon identified arnT , which encodes a transferase that adds aminoarabinose to lipid A in the OM [111]. This modification likely increases the OM stability and resilience to mechanical and osmotic pressure.…”