The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm

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.

1 Supplementary Figure 1. Multiple-sequence alignment of MlaC family proteins with known 3D structure based on a structural alignment. CATH domains 1 (D1) and 2 (D2), each with segments 1 (S1) and 2 (S2), are shown above the sequences together with their secondary structure elements. Identical residues are highlighted in red, similar residues are in red font and similar regions are boxed in blue. Residues highlighted in grey are missing in the PDB entries. Sequence numbers correspond to the P. aeruginosa protein (Ttg2DPae). Red stars indicate residues annotated in the binding site of MlaC from R. solanacearum (2QGU). Below the sequences, triangles indicate the first amino acid of the mature protein (after cleavage of the signal peptide) and squares the residues forming the cavities.  16 . Superposed residues are shown in thick Cα-trace. The table lists the RMSD values and the number of aligned residues for each structural alignment.   Figure 6. Superposition of the structure of Ttg2D from P. aeruginosa with known orthologous protein structures. Upper panels show superposition of Ttg2D crystal structures from P. aeruginosa (this study, PDB entry 6HSY), P. putida (5UWB), R. solanacearum (2QGU) and E. coli (5UWA) (stereo view). Same color coding as in Supplementary Fig. 1. Lower panels show superposition of the structures of Ttg2D from P. aeruginosa (green) and R. solanacearum (pink) in two different views. Residues that distinguish the group of proteins that we predict to bind two diacyl lipids are indicated with residue letter and number. Besides G195 and W196, side-chain orientation and hydrophobicity of the other indicated residues in these regions could be also contributing to a tighter binding of the two diacyl phospholipids inside the ligand cavity. Residues in region 65-83 are represented as ball-and-stick and residues in region 154-198 as sticks.  Fig. 3A). The most represented species are complexes of the protein (P) containing either one (1PL, deconvoluted MW=23551 Da) or two (2PL, deconvoluted MW=24296) phospholipid molecules. Above the spectrum, the inferred amino acid sequence for the recombinant protein is displayed, highlighting the 6xHis tail in yellow and other added amino acids from pET vector in blue. The SDS-PAGE shows the purified recombinant protein (first column: MW marker in kDa). The theoretical monoisotopic MW of the recombinant protein that has lost the first methionine is 22836.78 Da. Supplementary Figure 11. Fragmentations of isolated ion m/z=2430 (z=10) at varying transfer collision energies (CE) from the ligand-binding assays to study the interaction in vitro between delipidated Ttg2DPae (P) and cardiolipin (CL  2,3 . Numbers associated to each species indicate the number of carbon atoms and double bonds, respectively, for each fatty acid side chain. Fatty acid cyclopropylation (cy) is equivalent to unsaturation in terms of molecular mass. When it was not possible to assign the fatty acid species, the total number of carbon atoms and double bonds is shown in parentheses. # Lipidome analyses made for this work are shown in Supplementary Fig. 10. Column indicates whether PL was detected in these analyses.  4 . Numbers associated to each species indicate the number of carbon atoms and double bonds, respectively, for each fatty acid side chain. # Lipidome analyses made for this work are shown in Supplementary Fig. 10. Column indicates whether PL was detected in these analyses. Table 4. Antibiotic susceptibility profile of P. aeruginosa mutants of the Ttg2/VacJ system and complemented strains for Δttg2D. † Minimum inhibitory concentration (MIC) determined by the broth microdilution method except for polymyxin B that was determined by Etest (ND: not determined). MICs were confirmed by two or three independent replicates and MIC differences greater than 2-fold with respect to the wild type strain were considered significant (indicated with an asterisk). Transposon mutants of P. aeruginosa PAO1 and complemented strains are described in Supplementary Table 5.  This study † Genotype for UW mutants referenced in the following way: gene name-well name as the allele number::Transposon name. tetA, tetracycline-resistance gene. kan, kanamycin-resistance gene. All mutants contained either an ISlacZ/hah or an ISphoA/hah transposon insertion. Between parenthesis strain name at the UW mutant library [http://www.gs.washington.edu/labs/manoil/libraryindex.htm]. ‡ MPAO1 (PAO1 for short) was received from the distributor of the PAO1 mutant library of the University of Washington, Seattle 5 . Note:

Supplementary
Further information on UW mutants can be found at http://www.gs.washington.edu/labs/manoil/libraryindex.htm. The correct insertion of the transposon into the mutant strains was confirmed in the recent sequence-verified collection of UW mutants 6 and for mutants showing differential phenotypes the transposon location was also confirmed by colony PCR following the protocol and primers recommended by the University of Washington Genome Science Center. § The Liverpool epidemic strain (LES) B58, known as LESB58, was kindly donated by Dr. Roger C. Levesque, IBIS, Université Laval, Québec (Canada). ¶ P. aeruginosa C17 was isolated from an ICU patient at Hospital Clinic, Barcelona (Spain), upon screening of surveillance rectal swabs in August 2007. P. aeruginosa MDR strain PAR-10821 was also isolated at the Hospital Clinic in 2012.

Supplementary Methods
Bacterial growth conditions. Unless stated otherwise, strains were routinely cultured on Luria-Bertani broth (LB) agar plates, or to exponential phase (OD550 of 1.0), or up to late exponential phase (OD550 of 2.7 to 3.0) in LB at 37°C with shaking at 250 rpm. When necessary, antibiotics were added at final concentrations of 500 μg/ml for erythromycin, 10 μg/ml for gentamicin, 17 μg/ml for tetracycline, 5 μg/ml for norfloxacin, or 50 μg/ml for kanamycin for Escherichia coli or 1000 μg/ml for erythromycin, 40 μg/ml for gentamicin, or 60 μg/ml for tetracycline for Pseudomonas aeruginosa. Harvested crystals were directly flashed cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on the beamline ID23-1 at the European Synchrotron Radiation Facility (Grenoble, France) 18 . These data were indexed, integrated, scaled and merged using iMOSFLM 19 and AIMLESS 20 . Ttg2DPae structure was solved with Phaser 21 using a poly-alanine model built with MODELLER 22 from the homologous protein of Ralstonia solanacearum (PDB code 2QGU, 25% sequence identity). The structure was automatically re-built with one run of AutoBuild, followed by iterative cycles of restrained refinement with Phenix.refine 23 Table 5). Then, primers ttg2DcompF and ttg2DHiscompR (Supplementary Table 5) were used to amplify Ttg2D coding sequence including the signal sequence and the RBS, and the resulting amplicon inserted between sites NheI and HindIII into the new vector pBBR1-pBAD-Gm. Primer ttg2DHiscompR includes a sequence coding for six consecutive histidines. The resulting expression vector pBBR1-pBAD-ttg2DHis was introduced into P. aeruginosa PAO1 Δttg2D by electroporation 32 .
For protein production, P. aeruginosa mutant strain carrying plasmid pBBR1-pBAD-ttg2DHis was grown in BM2 glycerol medium with casamino acids [62 mM potassium phosphate buffer (pH 7.0), 5 mM MgSO4, 10 µM FeSO4, 0.5% casamino acids and 0.4% glycerol] supplemented with 40 μg/ml gentamicin. A bacterial inoculum from an overnight culture was diluted 1/100 in 1 l of fresh medium and grown to mid-exponential phase (OD550  0.6) at 37°C with vigorous shaking. Cells were centrifuged at 4000 g for 10 min at 4°C, washed twice in 30 mM Tris-HCl (pH 7.0) and 150 mM NaCl and they were kept in ice. The method of spheroplasting by lysozyme and sucrose 33  Trap gas flow was 1.5 ml/sec. The bias voltage for entering in the T-wave cell was 15 V.  Generation of ttg2 mutants in MDR P. aeruginosa strains using the pGPI-SceI/pDAI-SceI-SacB system. This mutagenesis method is based on the I-SceI homing endonuclease system, which relies on two independent crossover events to integrate first a deletion plasmid with a I-SceI recognition site into the genome of the recipient and then resolve the co-integrate structure by a second homologous recombination event in the presence of the I-SceI endonuclease provided in trans on a replicative plasmid 13,35 . One of these mutagenesis systems relies on an improved suicide vector that contains an I-SceI restriction site and the xylE reporter gene (pGPI-SceI-XCm), and a replicative but unstable plasmid that encodes the I-SceI endonuclease and the counterselectable marker SacB (pDAI-SceI-SacB) 11 . To successfully achieve genetic manipulations in MDR P. aeruginosa strains, we further modified pGPI-SceI-XCm by introducing an erythromycin resistance determinant replacing the chloramphenicol resistance cassette. To make this decision, the MIC for erythromycin was previously analyzed in our P. aeruginosa strains using the microdilution technique and found to be at a level of 256 μg/ml. The erythromycin resistance (erm) gene from plasmid pNZerm 36 was PCR amplified using primers Erm5'-PstI and Erm3'-PstI (Supplementary Table 5) and with pNZerm DNA as a template. The resulting amplicon (1026 bp) was digested with PstI and cloned into PstI-digested pGPI-SceI-XCm to create pGPI-SceI-XErm. Previously, the region between the SacI sites of plasmid pGPI-SceI-XCm (359 bp) was deleted by digestion with this restriction enzyme and ligation. This region contains the Pc promoter found in class 1 integrons (promoter for the trimethoprim resistance gene dhfrIIb in pGPI-SceI vectors) and this could cause unwanted integration of the suicide vector into the P. aeruginosa chromosome of some strains. Class 1 integrons have been detected with high prevalence in P. aeruginosa 37 .
The mutagenesis plasmid for the ttg2 operon deletion (from PA4452 to PA4456 in PAO1) was constructed by PCR amplification of DNA fragments flanking this gene cluster from PAO1 strain, which were cloned into pGPI-SceI-XErm (see Supplementary Table 5 for primer details). The upstream fragment (633 bp) was amplified using primers US'-ttg2-U and US'-ttg2-L. The downstream fragment (818 bp) was amplified using primers DS'-ttg2-U and DS'-ttg2-L. The upstream fragment was digested with EcoRI and NheI, the downstream fragment was digested with NheI and BglII, and both fragments were inserted in two successive cloning steps into pGPI-SceI-XErm to create pΔttg2-US'DS'. The successful construction of the mutagenesis plasmid was verified by DNA sequence analysis of the inserts. All deletion plasmids were generated and maintained in E. coli SY327.
The mutagenic plasmid pΔttg2-US'DS' was mobilized into P. aeruginosa C17, PAER-10821 and LESB58 by triparental mating 35 using E. coli DH5α carrying the plasmid pRK2013 as a helper strain. Modifications of the method include that the recipient P.
aeruginosa strains were incubated at 42°C overnight before conjugation because certain strains presumably contain restriction systems that could severely restrict foreign DNA.
Erythromycin at 1000 μg ml -1 was used to select for cointegrants (single-crossover clones) in these P. aeruginosa strains, and 5 μg/ml norfloxacin to counter-select against the E. coli helper and donor strains. To distinguish true cointegrants from colonies that spontaneously became resistant to erythromycin, streaks of exconjugants were sprayed with 0.45M pyrocatechol since in the presence of this compound colonies expressing 2,3-catecholdioxygenase encoded by xylE turned bright yellow 38 . For the final mutagenesis stage, pDAI-SceI-SacB was mobilized into P. aeruginosa cointegrants, and exconjugants were selected on LB agar plates containing 60 μg/ml tetracycline and 5 μg/ml norfloxacin for PAER-10821 and LESB58 derivatives. Tetracycline-resistant colonies, appearing after 48 hours, were screened by PCR and sequencing to confirm the deletion using the primers Ext-ttg2-U and Ext-ttg2-L (Supplementary Table 5) that anneal to sequences outside the deleted region. Detection of deletion mutants cured from the plasmid pDAI-SceI-SacB was achieved by growing P. aeruginosa on LB plates without salt and supplemented with 5% (wt/vol) sucrose and then screening the resulting colonies for loss of tetracycline resistance. To obtain a ttg2 mutant in the P. aeruginosa C17 strain, the derivative cointegrates have to be resolved by spontaneous recombination of the allele pair since resolution via the I-SceI endonuclease provided on the plasmid pDAI-SceI-SacB did not work. Failure during the second recombination event for C17 strain (resolution always restores the parental allele) was probably due to the mutation in the ttg2 operon making the cells more susceptible to tetracycline 39 , the antibiotic resistance marker to select for cells carrying plasmid pDAI-SceI-SacB. In this case, resolution of cointegrates was achieved by plating several thousand colonies on LB plates without selection. For this, the single-crossover clones were serially subcultured in LB without selection for two consecutive days and then diluted up to 10 -8 prior to spread over the plates. Plates were sprayed with 0.45 M pyrocatechol to screen for xylE negative cells (no yellow coloration).
The screening was facilitated because P. aeruginosa xylE negative colonies turned dark brown after spraying with pyrocatechol ( Supplementary Fig. 13). Selected colonies were screened by PCR to confirm the deletion using the primers Ext-ttg2-U and Ext-ttg2-L.

Vectors for complementation.
For complementation purposes of the PAO1 derivative mutant strains, the broad-host-range cloning vectors pBBR1MCS-5 14 or its derivative variants pBBR1-pBAD-Gm (see Ttg2D homologous expression in P. aeruginosa) or pBBR1MCS-6 were used with the oligonucleotide primers described in Supplementary  Table 5) and with pNZerm DNA as a template. The resulting amplicon (1026 bp) was digested with KpnI and BglII and cloned into KpnI/BglII -digested pBBR1MCS-5 to create pBBR1MCS-6. The full ttg2 operon (3650 bp) from P. aeruginosa PAO1 was cloned separately into pBBR1MCS-5 or pBBR1MCS-6 as described in Supplementary Table 5. The complementation vectors were introduced into P. aeruginosa by electroporation 32 . Outer membrane permeabilization assay.

Ttg2/Mla pathway and resistance to antimicrobial agents
The proposed function of the Ttg2/Mla pathway in membrane remodeling provides a plausible explanation for the pleiotropic resistance phenotypes shown by the ttg2 mutants in this study, including resistance to various antibiotics, chelating agents and organic solvents. In addition, these mutations increase the deleterious effect of antibiofilm agents like EDTA, a substance with known low activity against biofilms of P. aeruginosa PAO1 53 .
Mutations in orthologous ttg2 genes in other Gram-negative organisms have been shown to affect diverse physiological processes, mainly associated with an increased OM permeability. In E. coli, the mutants defective in components of the Mla system rendered cells more susceptible to the lethal action of quinolones, the detergent SDS and EDTA 54,55 . Mutants for the orthologs of the Ttg2 pathway in both Shigella flexneri and Francisella novicida resulted also in increased sensitivity to lysis by SDS 56,57 . In addition, in S. flexneri this pathway appears to play a role in the intercellular spread of the bacteria between adjacent epithelial cells 56 . In fact, the Ttg2/Mla pathway has proven to be an important virulence factor in other pathogens, like Burkholderia pseudomallei, that need to spread into neighboring cells to infect eukaryotic tissues 58 . In Burkholderia cepacia complex species, mla genes are required for swarming motility and serum resistance 59 .
Furthermore, in nontypeable Haemophilus influenzae (NTHi), it is considered a key factor for bacterial survival in the human airway upon exposure to hydrophobic antibiotics 60 . In S.
flexneri, B. pseudomallei and NTHi the role of the mla operon in virulence has been inferred from mutants for the gene vacJ (mlaA) 58,61 . This gene is predicted to be part of the Ttg2 ABC transport system, since it is found in an operon with ttg2 homologs in other bacteria 62 . In agreement with our work, it has been previously shown that in P. aeruginosa VacJ plays an important role in both virulence and antibiotic susceptibility to ciprofloxacin, chloramphenicol and tetracycline 63 .
Colistin is considered a last-resort antibiotic for the treatment of infections by several MDR Gram-negative pathogens, but its use against MDR P. aeruginosa is increasingly impeded by colistin resistance 64 . A variety of gene mutations are known to cause resistance to colistin by altering the OM of Gram-negative bacteria, for example, by covalent modification of the lipid A constituent of LPS as consequence of mutations in the PhoPQ two component regulatory system 65,66 . In P. aeruginosa, the PhoPQ system plays a role in the induction of resistance to polymyxins in response to limiting divalent cations, as well as in virulence 67,68 , and this system has been recently identified as a regulator of P.
aeruginosa's ttg2 operon 39 . Interestingly, in Salmonella the increase in OM cardiolipins is regulated by PhoPQ and it is necessary for their virulence 69 . More recently, nucleotide polymorphisms conferring resistance to polymyxins have been detected in genes of the Mla pathway in A. baumannii 70 . Although data on the precise mechanisms of resistance are scant and appear to be dependent on specific regulatory systems 67,71 , the activity of the Ttg2 system on membrane phospholipid homeostasis appears to be partly responsible for the lower basal susceptibility of P. aeruginosa to colistin.