Resistance to the Antimicrobial Peptide Polymyxin Requires Myristoylation of Escherichia coli and Salmonella typhimurium Lipid A
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m505020200
ISSN1083-351X
AutoresAn X. Tran, Melissa E. Lester, Christopher M. Stead, Christian R.H. Raetz, Duncan J. Maskell, Sara C. McGrath, Robert J. Cotter, M. Stephen Trent,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoAttachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrAC strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3′-acyloxyacyl-linked myristate (C14) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group. Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrAC strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3′-acyloxyacyl-linked myristate (C14) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-d-manno-octulosonic acid; l-Ara4N, 4-amino-4-dexoy-l-arabinose; pEtN, phosphoethanolamine; ArnT, l-4-aminoarabinose transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. is the major surface molecule of Gram-negative bacteria and is held in the outer membrane by a unique phospholipid domain known as lipid A. The typical lipid A backbone consists of a β-1′,6-linked disaccharide of glucosamine that is phosphorylated and multiply acylated. In Escherichia coli and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), the disaccharide backbone is acylated at the 2-, 3-, 2′-, and 3′-positions with (R)-3-hydroxymyristate and phosphorylated at the 1- and 4′-positions (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). A secondary lauroyl (C12) and myristoyl (C14) group is attached at the 2′- and 3′-positions, respectively, of the distal glucosamine in an acyloxyacyl linkage resulting in the hexa-acylated structure shown in Fig. 1A (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). The lipid A domain is attached to the polysaccharide portion of LPS via the Kdo (3-deoxy-d-manno-octulosonic acid) sugars (Fig. 1) (1.Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar). Modification of the lipid A domain of E. coli and S. typhimurium with the cationic sugar 4-amino-4-dexoy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) promotes resistance to the cyclic antimicrobial lipopeptide, polymyxin (2.Helander I.M. Kilpelainen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, 3.Nummila K. Kilpelainen I. Zahringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (174) Google Scholar, 4.Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (245) Google Scholar, 5.Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar). Although the mechanism of polymyxin killing is not completely understood, the peptide is thought to access the outer surface of the bacterium by interacting with the negatively charged phosphate groups of lipid A. A similar mechanism is employed by cationic antimicrobial peptides of the innate immune system (6.Vaara M. Microbiol. Rev. 1992; 56: 395-411Crossref PubMed Google Scholar). Masking of lipid A phosphate groups with positively charged amine-containing residues is predicted to decrease binding of polymyxin to the bacterial surface promoting survival. In E. coli and S. typhimurium, the polymyxin-resistant phenotype is primarily under the control of the PmrA/PmrB two-component regulatory system that is activated during growth under conditions of low pH, high Fe3+, and in a PhoP/PhoQ-dependent manner during Mg2+ starvation (7.Wosten M.M. Kox L.F. Chamnongpol S. Soncini F.C. Groisman E.A. Cell. 2000; 103: 113-125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 8.Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (340) Google Scholar, 9.Groisman E.A. J. Bacteriol. 2001; 183: 1835-1842Crossref PubMed Scopus (658) Google Scholar). Previously, Trent and co-workers (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) demonstrated that periplasmic addition of l-Ara4N to lipid A is catalyzed by l-4-aminoarabinose transferase (ArnT), a PmrA-regulated glycosyltransferase (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Because the transferase utilizes an undecaprenyl-linked donor substrate, undecaprenyl-phosphate-α-l-Ara4N, its active site is predicted to lie in the periplasmic region of the cell (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Furthermore, Doerrler et al. (12.Doerrler W.T. Gibbons H.S. Raetz C.R. J. Biol. Chem. 2004; : 45102-45109Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) have demonstrated that modification of lipid A with l-Ara4N and pEtN is dependent upon its transport across the inner membrane by MsbA. We now report that, in E. coli K12 and S. typhimurium, addition of l-Ara4N to the lipid A domain of LPS in living cells is dependent upon the presence of the acyloxyacyl-linked myristoyl group at the 3′-position. Loss of myristoylation of lipid A in both E. coli and S. typhimurium by inactivation of lpxM resulted in loss of l-Ara4N modification and in a significant decrease in polymyxin resistance. However, the pEtN modification of the lipid A phosphate groups was not effected by loss of myristoylation. Chemicals and Other Materials—[γ-32P]ATP and 32Pi were obtained from Amersham International. Silica Gel 60 (0.25-mm) thin layer plates were purchased from EM Separation Technology (Merck). Yeast extract and Tryptone were from Difco. Triton X-100 and bicinchoninic acid were from Pierce. Polymyxin B sulfate was purchased from Sigma. All other chemicals were reagent grade and were purchased from either Sigma or Mallinckrodt. Bacterial Strains and Growth Conditions—Bacterial strains are described in Table I. Typically bacteria were grown at 37 °C in LB broth containing 10 g of NaCl, 10 g of Tryptone, and 5 g of yeast extract per liter. When required for plasmid selection, cells were grown in the presence of 100 μg/ml ampicillin, 12 μg/ml tetracycline, 30 μg/ml chloramphenicol, or 30 μg/ml kanamycin.Table IStrains and plasmidsStrain or plasmidDescriptionSource or Ref.E. coliW3110Wild type, F-, λ-E. coli Genetic Stock Center, Yale UniversityWD101W3110, pmrAc, polymyxinr11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google ScholarMLK1067W3110, lpxM::Ωcam13.Clementz T. Zhou Z. Raetz C.R. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 14.Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google ScholarWD103WD101, lpxM::ΩcamThis workF2-1WD101 polymyxins mutantThis workC1-1WD101 polymyxins mutantThis workA3-1WD101 polymyxins mutantThis workS. typhimuriumC5Wild type mouse virulent strain28.Khan S.A. Everest P. Servos S. Foxwell N. Zahringer U. Brade H. Rietschel E.T. Dougan G. Charles I.G. Maskell D.J. Mol. Microbiol. 1998; 29: 571-579Crossref PubMed Scopus (176) Google ScholarC5 lpxM::kanKanr28.Khan S.A. Everest P. Servos S. Foxwell N. Zahringer U. Brade H. Rietschel E.T. Dougan G. Charles I.G. Maskell D.J. Mol. Microbiol. 1998; 29: 571-579Crossref PubMed Scopus (176) Google ScholarC5 lpxM::kan/pR1AKanr,AmprThis workC5 lpxM::kan/pRM17AKanr,AmprThis workPlasmidspR1ADerivative of pACYC177, AmprThis workpRM17ApR1A containing S. typhimurium lpxMThis workpWSK29Low-copy expression vector, Ampr15.Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1013) Google ScholarpWSLpxMpWSK29 containing E. coli lpxMThis work Open table in a new tab Recombinant DNA Techniques—Plasmids were isolated using the Qiagen Spin Prep Kit. Custom primers were obtained from Integrated DNA Technologies. PCR reagents were purchased from Stratagene. PCR clean up was performed using the Qiaquick PCR Purification Kit (Qiagen). DNA fragments were isolated from gels using the Qiaquick Gel Extraction Kit (Qiagen). Restriction endonucleases, T4 DNA ligase, and shrimp alkaline phosphatase were purchased from New England Biolabs. All modifying enzymes were used according to the manufacturer's instructions. Isolation of Polymyxin-sensitive Mutants—Polymyxin-sensitive mutants were generated by random mutagenesis of the E. coli strain, WD101. WD101, a polymyxin-resistant E. coli K12 strain, contains a mutation in the pmrA (basR) gene resulting in a pmrAC phenotype promoting polymyxin resistance (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). E. coli WD101 was treated with 40 μg/ml of the mutagen N-methyl-N′-nitro-N-nitrosoguanidine for 10 min at 37 °C. To obtain clones that were sensitive to polymyxin, mutagenized cells were first grown on LB agar plates (∼100/plate). Sensitive mutants were identified by replica plating onto LB agar plates containing 2 μg/ml polymyxin B sulfate at 37 °C. Sensitive clones were re-purified and their sensitivity to polymyxin verified. Approximately 10,000 colonies were screened by the above method, and ∼100 polymyxin-sensitive mutants were identified including strains F2-1 and C1-1. Generation of E. coli Strain WD103—WD103 was generated by P1vir transduction of the polymyxin-resistant genotype (pmrAC) of strain WD101 into the E. coli lpxM mutant MLK1067 (W3110 lpxM::Ωcam) (13.Clementz T. Zhou Z. Raetz C.R. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 14.Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). Construction of LpxM Covering Plasmids, pWSLpxM and pRM17A— E. coli lpxM was cloned into the multiple cloning site of the low-copy expression vector pWSK29 (15.Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1013) Google Scholar) resulting in plasmid pWSLpxM. The latter was used to complement the pmrAC E. coli lpxM mutant, WD103. The S. typhimurium lpxM covering plasmid was constructed by subcloning the Salmonella lpxM gene into a pACYC derivative, pR1A. The resulting plasmid was named pRM17A and was used to complement the S. typhimurium lpxM mutant. Isolation and Analysis of 32P-Labeled Lipid A Species—32Pi-Labeled lipid A was isolated from cells uniformly labeled with 2.5 μCi/ml of 32Pi in 5 ml of LB broth as previously described (16.Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The 32P-labeled lipid A domain was released from LPS by hydrolysis of the Kdo dissacharide and processed by the method of Zhou and co-workers (16.Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Lipids were spotted onto a Silica Gel 60 TLC plate (10,000 cpm/lane) and developed in the solvent chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). Labeled lipids were visualized by phosphorimaging. Isolation and Analysis of 32P-Labeled Undecaprenyl-Phosphate-α-l-Ara4N—Cells were uniformly labeled with 32Pi as described above. The glycerolphospholipid fraction containing the undecaprenyl-phosphate-α-l-Ara4N was isolated and analyzed by TLC as described by Trent and co-workers (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Preparation of Cell-free Extracts, Double-Spun Cytosol, and Washed Membranes—Typically, 200-ml cultures of E. coli or S. typhimurium were grown at 37 °C to an A600 of 1.0 and harvested by centrifugation at 6,000 × g for 30 min. All samples were prepared at 4 °C. Cell-free extract, double-spun cytosol, and washed membranes were prepared as previously described (17.Trent M.S. Pabich W. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and were stored in aliquots at –20 °C. Protein concentration was determined by the bicinchoninic acid method (18.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar), using bovine serum albumin as the standard. Preparation of [4′-32P]Lipid IVA—The substrate [4′-32P]lipid IVA was generated from 100 μCi of [γ-32P]ATP and the tetraacyldisaccharide 1-phosphate lipid acceptor, using the overexpressed 4′-kinase present in membranes of E. coli BLR(DE3)/pLysS/pJK2 (17.Trent M.S. Pabich W. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 19.Garrett T.A. Kadrmas J.L. Raetz C.R. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 20.Basu S.S. York J.D. Raetz C.R. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), as previously described (17.Trent M.S. Pabich W. Raetz C.R. Miller S.I. J. Biol. Chem. 2001; 276: 9083-9092Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 20.Basu S.S. York J.D. Raetz C.R. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Assay of E. coli ArnT—The ArnT was assayed as previously described by Trent and co-workers using [4′-32P]lipid IVA as the substrate (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Addition of the l-Ara4N was visualized as a shift to a slower migrating lipid species previously termed Lipid IIA (21.Raetz C.R. Purcell S. Meyer M.V. Qureshi N. Takayama K. J. Biol. Chem. 1985; 260: 16080-16088Abstract Full Text PDF PubMed Google Scholar). Polymyxin Sensitivity Assays—E. coli and S. typhimurium strains were assayed for polymyxin resistance as described by Gunn and Miller (8.Gunn J.S. Miller S.I. J. Bacteriol. 1996; 178: 6857-6864Crossref PubMed Scopus (340) Google Scholar). After growth to an A600 nm of ∼0.5, bacteria were diluted to ∼2,500 colony forming units/ml in LB broth. Cells (200 μl) were treated for 1 h at 37 °C with increasing concentrations of polymyxin in a microtiter plate. Following exposure to polymyxin, 100 μl of treated bacteria were plated on LB agar plates and incubated overnight at 37 °C. Colony counts were determined for each concentration of polymyxin tested, and the results were expressed as the percentage of colonies resulting from untreated cells. Large Scale Isolation of Lipid A—E. coli or S. typhimurium were cultured in 200 ml of LB medium at 37 °C or N-minimal media containing 10 μm Mg2+, respectively. The cultures were allowed to reach an A600 nm ∼ 0.8, harvested by centrifugation at 6000 × g for 15 min, and washed once with phosphate-buffered saline. The final cell pellets were resuspended in 20 ml of phosphate-buffered saline. Lipid A was released from cells and purified as previously described (16.Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 22.Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and stored frozen at –20 °C. Prior to mass spectrometry various lipid A species were separated by anion-exchange chromatography as previously described (22.Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 37.Strain S.M. Armitage I.M. Anderson L. Takayama K. Qureshi N. Raetz C.R. J. Biol. Chem. 1985; 260: 16089-16098Abstract Full Text PDF PubMed Google Scholar). Mass Spectrometry of Lipid A Species—Mass spectra of the purified lipids were acquired in the negative ion mode using a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometer (AXIMA-CFR, Kratos Analytical, Manchester, UK), equipped with a nitrogen laser (337 nm). The instrument was operated using 20-kV extraction voltage and time-delayed extraction, providing a mass resolution of about ±1 atomic mass units for compounds with Mr ∼ 2000. Each spectrum represented the average of 100 laser shots. Saturated 6-aza-2-thiothymine in 50% acetonitrile and 10% tribasic ammonium citrate (9:1, v/v) served as the matrix. The samples were dissolved in chloroform/methanol (4:1, v/v), and deposited on the sample plate followed by an equal portion of matrix solution (0.3 μl). The sample was dried at 25 °C prior to mass analysis. Isolation and Characterization of Polymyxin-sensitive Mutants of E. coli WD101—Addition of the amine-containing residues, l-Ara4N and pEtN, to the phosphate groups of lipid A (Fig. 1) is associated with increased resistance to cationic antimicrobial peptides, including the peptide polymyxin (2.Helander I.M. Kilpelainen I. Vaara M. Mol. Microbiol. 1994; 11: 481-487Crossref PubMed Scopus (146) Google Scholar, 3.Nummila K. Kilpelainen I. Zahringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (174) Google Scholar, 4.Lee H. Hsu F.F. Turk J. Groisman E.A. J. Bacteriol. 2004; 186: 4124-4133Crossref PubMed Scopus (245) Google Scholar, 5.Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar). E. coli strain WD101 contains a mutation in the pmrA gene resulting in a pmrAC phenotype and modification of the lipid A structure with l-Ara4N and pEtN, helping to provide resistance to polymyxin at concentrations of up to 20 μg/ml on solid media (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Originally, we sought to determine additional modifications to the lipid A structure that were important for polymyxin resistance. Using WD101 as the parent strain, a library of mutants displaying sensitivity to polymyxin at 2 μg/ml was generated via random chemical mutagenesis. Approximately 10,000 colonies were replica plated on LB agar containing 2 μg/ml of polymyxin, and ∼100 colonies showing sensitivity to the peptide were purified for further analysis. Each mutant was uniformly labeled in the presence of 32Pi, and the lipid A fraction analyzed by TLC. Fig. 2 shows the modification patterns found in the lipid A fractions isolated from selected polymyxin-sensitive mutants. Overall, four different lipid A phenotypes were found among the polymyxin-sensitive mutants. For example, some mutants produced lipid A identical to that of wild type E. coli strain W3110. W3110 synthesizes a bis-phosphorylated hexa-acylated lipid A species lacking modified phosphate groups (Fig. 2, lane 1) and shows sensitivity to polymyxin at concentrations <0.1 μg/ml on solid media (data not shown). Mutant B1-1 produced lipid A species (Fig. 2, lane 4) identical to those of the WD101 parent strain (Fig. 2, lane 2) modified with l-Ara4N, and pEtN yet showed sensitivity to the peptide at 2 μg/ml. These results suggest that other mechanisms involved in conferring polymyxin resistance to E. coli may remain as yet unidentified. As expected, in some cases sensitivity arose from the inability to synthesize l-Ara4N- or pEtN-modified species. For example, strain A1-1 (Fig. 2, lane 3) produced lipid A modified with one or two pEtN residues, whereas mutant D1-1 produced species modified with only l-Ara4N (lane 6). The changes in the lipid A domain of A1-1 and D1-1 most likely arose from lack of l-Ara4N or pEtN transferase activities, respectively (data not shown). Finally, strains C1-1 and F2-1 produced very hydrophilic lipid A species that had not been previously identified, and for this reason these strains were chosen for further analysis. Strains C1-1 and F2-1 Have the Enzymatic Machinery to Synthesize and Transfer l-Ara4N to Lipid A—Transfer of l-Ara4N to lipid A occurs in the periplasmic region of the cell catalyzed by the inner membrane glycosyltransferase, ArnT. The enzyme utilizes an undecaprenyl carrier lipid, undecaprenyl-phosphate-α-l-Ara4N, as the donor substrate (10.Trent M.S. Ribeiro A.A. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43122-43131Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Loss of ArnT function or the inability to synthesize the undecaprenyl-linked substrate results in loss of polymyxin resistance (5.Gunn J.S. Lim K.B. Krueger J. Kim K. Guo L. Hackett M. Miller S.I. Mol. Microbiol. 1998; 27: 1171-1182Crossref PubMed Scopus (508) Google Scholar, 23.Gunn J.S. Ryan S.S. Van Velkinburgh J.C. Ernst R.K. Miller S.I. Infect. Immun. 2000; 68: 6139-6146Crossref PubMed Scopus (315) Google Scholar). Because mutants C1-1 and F2-1 showed sensitivity to polymyxin, we determined if these mutants contained the necessary enzymatic machinery to synthesize and transfer l-Ara4N to the lipid A domain of their LPS. The phospholipid fraction from 32Pi-labeled cells was analyzed by TLC for the presence of the undecaprenyl-phosphate-α-l-Ara4N donor lipid. As previously shown by Trent et al. (11.Trent M.S. Ribeiro A.A. Doerrler W.T. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2001; 276: 43132-43144Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), the l-Ara4N donor lipid was produced by the polymyxin-resistant strain WD101 (Fig. 3A, lane 1), but absent in the polymyxin-sensitive wild type strain, W3110 (lane 2). However, examination of the phospholipid fraction from mutants C1-1 and F2-1 (Fig. 3A, lanes 3 and 4) showed the presence of the l-Ara4N donor lipid. Furthermore, mutants C1-1 and F2-1 contained functional ArnT. Membranes isolated from either C1-1 or F2-1 catalyzed the transfer of l-Ara4N from the donor substrate to [4′-32P]lipid IVA (Fig. 3B, lanes 4 and 5), a tetra-acylated bis-phosphorylated precursor of lipid A. From this data we were able to conclude that a decrease in polymyxin resistance in strains C1-1 and F2-1 did not arise from the inability to produce or transfer l-Ara4N to lipid A. Analysis of Lipid A Produced by Polymyxin-sensitive Mutants C1-1 and F2-1 by MALDI-TOF Mass Spectrometry—The lipid A species of mutants C1-1 or F2-1 were isolated as previously described and separated based upon charge using anion-exchange chromatography (16.Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 22.Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Mass spectrometry of the lipid A species containing unmodified phosphate groups revealed the presence of a penta-acylated bis-phosphorylated species as indicated by [M-H]– at m/z 1587.6 in the negative mode. The major ion peak at 1587.6 (Fig. 4A) corresponds to the loss of myristate (C14) from E. coli lipid A that is found in E. coli mutants lacking a functional myristoyltransferase, LpxM (MsbB) (24.Vorachek-Warren M.K. Ramirez S. Cotter R.J. Raetz C.R. J. Biol. Chem. 2002; 277: 14194-14205Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 25.Somerville Jr., J.E. Cassiano L. Bainbridge B. Cunningham M.D. Darveau R.P. J. Clin. Investig. 1996; 97: 359-365Crossref PubMed Scopus (242) Google Scholar). The majority of the penta-acylated lipid was modified with either one or two pEtN groups as indicated by major ion peaks at m/z 1710.6 (Fig. 4A) and 1832.6 (Fig. 4B), respectively. Interestingly, mutants C1-1 or F2-1 were unable to produce lipid A modified with l-Ara4N even though both mutants contain the necessary enzymatic machinery required for transfer of l-Ara4N to lipid A (see Fig. 3). The only additional modification present was addition of palmitate (C16) to species containing either one or two pEtN groups producing minor ion peaks at m/z 1948.9 and 2071.4, respectively (Fig. 4). Addition of the palmitoyl group is catalyzed by the outer membrane enzyme, PagP (26.Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (282) Google Scholar, 27.Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). This data were suggestive that the increased polymyxin sensitivity seen displayed by mutants C1-1 or F2-1 resulted from a loss of l-Ara4N addition. Second, these results suggest that myristoylation of E. coli lipid A in vivo is a requirement for l-Ara4N addition. Generation and Characterization of a pmrAC, lpxM::Ωcam E. coli Mutant—To determine whether loss of l-Ara4N modification in mutants C1-1 and F2-1 arose from a loss of addition of myristate to lipid A, we constructed the E. coli strain WD103. A P1vir bacteriophage lysate of polymyxin-resistant WD101 was used to transduce the pmrAC mutation into an existing E. coli lpxM (msbB) mutant, MLK1067 (13.Clementz T. Zhou Z. Raetz C.R. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 14.Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). The resulting strain was referred to as WD103. Cultures of the various mutants and their corresponding parent strains were labeled with 32Pi, and the lipid A fraction isolated to determine possible modifications in vivo. As expected, the polymyxin-sensitive parent strains MLK1067 (Fig. 5, lane 3) and W3110 (Fig. 5, lane 1) produced unmodified penta- or hexa-acylated lipid A, respectively. The lipid A species shown in lane 5 isolated from WD103 (pmrAC, lpxM::Ωcam) were found to migrate with identical RF values of those isolated from mutants C1-1 (Fig. 5, lane 4) or F2-1 (data not shown). Complementation of strain WD103 with a low-copy vector expressing E. coli lpxM, pWSLpxM, resulted in production of lipid A identical to that found in the polymyxin-resistant parent strain WD101 (Fig, 5, lane 7). This was not the case when WD103 was complemented with the empty vector control, pWSK29 (lane 6). Analysis of the lipid A fraction of E. coli WD103/pWSK29 by MALDI-TOF mass spectrometry produced results identical to those shown in Fig. 4 for the polymyxin-sensitive mutant C1-1 (data not shown). WD103/pWSK29 produced pEtN-modified lipid A lacking both myristate and l-Ara4N. However, the lipid A of WD103/pWSLpxM produced major ions at m/z 1929.7, 2052.7, and 2290.7, corresponding to hexa-acylated lipid A modified with l-Ara4N (Fig. 5B). Table II provides a summary of the lipid A species of strains C1-1 and WD103/pWSLpxM identified by mass spectrometry.Table IIStructural interpretation of lipid A species detected by MALDI/TOF mass spectrometryStrainLipid A modificationsExpected [M-H]-Observed [M-H]-L-Ara4NpEtNC16-0C14-02-OHE. coli C1-1aSimilar data were obtained for E. coli strains F2-1 and WD103.000001588.01587.6010001711.11709.7/1710.6020001834.11832.6011001949.51948.9021002072.52071.4E. coli WD103/pLpxM000101798.41798.3100101929.51929.7110102052.52052.7111102290.92290.7S. typhimurium lpx
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