Abstract
In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.
Highlights
In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane
The available three-dimensional (3D) structures for the MlaC family from Ralstonia solanacearum (PDB entry 2QGU), P. putida (PDB entries 4FCZ and 5UWB), and E. coli (PDB entry 5UWA) were all solved in complex with a ligand in their hydrophobic pocket, except for one structure from E. coli (PDB entry 6GKI) where the protein was delipidated
A sequence alignment shows that some of the residues thought to be involved in phospholipid binding in the R. solanacearum Ttg2D structure are conserved in the P. aeruginosa ortholog (Supplementary Fig. 1)
Summary
In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg[2] system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes. A number of effective drugs and formulations can treat P. aeruginosa infections, even in CF patients[5] These include frontline antibiotics such as piperazillin-tazobactam, ceftazidime, aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, tobramycin, amikacin, and colistin[6]. The basis for the inherently high resistance of these microorganisms is primarily their low outer-membrane (OM) permeability[8,9], complemented by the production of antibiotic-inactivating enzymes (e.g. β-lactamases), the constitutive expression of efflux pumps[10,11], and the capacity to form biofilms[1,12], among other mechanisms. P. aeruginosa, the complex mechanisms controlling the basal, low-level resistance to these compounds are still poorly understood[16,17]
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