RESUMO
A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.
Assuntos
Amidoidrolases , Inibidores Enzimáticos , Escherichia coli , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Lipopolissacarídeos/biossíntese , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Membrana Celular/efeitos dos fármacosRESUMO
The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein-protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein-protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.
Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Amidoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
The outer membrane (OM) protects Gram-negative bacteria from harsh environmental conditions and provides intrinsic resistance to many antimicrobial compounds. The asymmetric OM is characterized by phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Previous reports suggested an involvement of the signaling nucleotide ppGpp in cell envelope homeostasis in Escherichia coli. Here, we investigated the effect of ppGpp on OM biosynthesis. We found that ppGpp inhibits the activity of LpxA, the first enzyme of LPS biosynthesis, in a fluorometric in vitro assay. Moreover, overproduction of LpxA resulted in elongated cells and shedding of outer membrane vesicles (OMVs) with altered LPS content. These effects were markedly stronger in a ppGpp-deficient background. We further show that RnhB, an RNase H isoenzyme, binds ppGpp, interacts with LpxA, and modulates its activity. Overall, our study uncovered new regulatory players in the early steps of LPS biosynthesis, an essential process with many implications in the physiology and susceptibility to antibiotics of Gram-negative commensals and pathogens.