Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

Abstract

22In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding 23 component of an ABC transport system thought to be involved in maintaining the 24 asymmetry of the outer membrane. The crystallographic structure of Ttg2D at 2.5Å 25 resolution reveals that this protein can bind two diacyl phospholipids. Native and 26 denaturing mass spectrometry experiments confirm that Ttg2D binds two 27 phospholipid molecules, which may have different head groups. Analysis of the 28 available structures of Ttg2D orthologs allowed us to classify this protein family as a 29 novel substrate-binding protein fold and to venture the evolutionary events that 30 differentiated the orthologs binding one or two phospholipids. In addition, gene 31 knockout experiments in P. aeruginosa PAO1 and multidrug-resistant strains show 32 that disruption of this system leads to outer membrane permeabilization. This 33 demonstrates the role of this system in low-level intrinsic resistance against certain 34 antibiotics that use a lipid-mediated pathway to permeate through membranes. 35 36 Introduction 37Pseudomonas aeruginosa are amongst the most important multidrug-resistant (MDR) 38 human pathogens 1 , showing inherent resistance to an important number of the 39 presently available antibiotics 2 . P. aeruginosa are responsible for chronic lung 40 infections in individuals with chronic obstructive pulmonary disease or cystic fibrosis 41 (CF) 3 and account for over a tenth of all nosocomial infections 4 . A number of effective 42 drugs and formulations can treat P. aeruginosa infections, even in CF patients 5 . 43These include frontline antibiotics such as piperazillin-tazobactam, ceftazidime, 44 aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, tobramycin, amikacin, 45 and colistin 6 . Yet, resistance to most of these antimicrobials is being increasingly 46 reported 7 . The basis for the inherently high resistance of these microorganisms is 47primarily their low outer-membrane (OM) permeability 8, 9 , complemented by the 48 production of antibiotic-inactivating enzymes (e.g. β-lactamases), the constitutive 49 expression of efflux pumps 10, 11 and the capacity to form biofilms 1, 12 , among other 50 mechanisms. The susceptibility of P. aeruginosa to antimicrobials can be additionally 51 reduced by the acquisition of inheritable traits, including horizontal gene transfers 52 and mutations that decrease uptake and efflux pump overexpression 13, 14, 15 . 53Although a number of genes and mechanisms of resistance to antibiotics are already 54 known in P. aeruginosa, the complex mechanisms controlling the basal, low-level 55 resistance to these compounds are still poorly understood 16, 17 . 56 57The OM of P. aeruginosa is known to be central to its antibiotic-resistance 58 phenotype. Its intrinsically low permeability is partly determined by inefficient OM 59 porin proteins that provide innate resistance to several antimicrobial compounds, 60 mainly hydrophilic 1, 8, 10 . On the other hand, the loss of specific efflux pump ...