Impact of the cAMP-cAMP Receptor Protein Regulatory Complex on Lipopolysaccharide Modifications and Polymyxin B Resistance in Escherichia coli.

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA, fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance.

Diacylglycerol Kinase A Is Essential for Polymyxin Resistance Provided by EptA, MCR-1, and Other Lipid A Phosphoethanolamine Transferases.

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated posttranscriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis. IMPORTANCE For Gram-negative bacteria, modification of a key surface structure known as lipopolysaccharide (LPS) is critical for resistance to cationic antimicrobial peptides, including the last-resort antibiotic polymyxin. One key enzyme that is critical for resistance is EptA that adds a positively charged residue to LPS, preventing polymyxin binding. Here we show that EptA can be posttranscriptionally regulated by a key cell envelope lipid leading to changes in antibiotic resistance.

Unsaturation Elements and Other Modifications of Phospholipids in Bacteria: New Insight from Ultraviolet Photodissociation Mass Spectrometry.

Glycerophospholipids (GPLs), one of the main components of bacterial cell membranes, exhibit high levels of structural complexity that are directly correlated with biophysical membrane properties such as permeability and fluidity. This structural complexity arises from the substantial variability in the individual GPL structural components such as the acyl chain length and headgroup type and is further amplified by the presence of modifications such as double bonds and cyclopropane rings. Here we use liquid chromatography coupled to high-resolution and high-mass-accuracy ultraviolet photodissociation mass spectrometry for the most in-depth study of bacterial GPL modifications to date. In doing so, we unravel a diverse array of unexplored GPL modifications, ranging from acyl chain hydroxyl groups to novel headgroup structures. Along with characterizing these modifications, we elucidate general trends in bacterial GPL unsaturation elements and thus aim to decipher some of the biochemical pathways of unsaturation incorporation in bacterial GPLs. Finally, we discover aminoacyl-PGs not only in Gram-positive bacteria but also in Gram-negative C. jejuni, advancing our knowledge of the methods of surface charge modulation that Gram-negative organisms may adopt for antibiotic resistance.