Altered lipid composition in Streptococcus pneumoniae cpoA mutants

BMC Microbiology

Regine Hakenbeck¹  Bernhard Henrich¹  Patrick Maurer²  Jessica Eisel¹  Carsten Volz³  Marina Meiers¹ 

[1]Department of Microbiology, University of Kaiserslautern, Gottlieb-Daimler-Strasse, Gebäude 23, D-67663 Kaiserslautern, Germany

[2]Present address: Hochschule für Technik und Wirtschaft des Saarlandes, Goebenstrasse 40, D-66117 Saarbrücken, Germany

[3]Present address: Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, D-66123 Saarbrücken, Germany

关键词: Phospholipid;    CpoA;    Glycosyltransferase;    Penicillin resistance;    Glycolipids;    Streptococcus pneumoniae;   

Others  :  1142183
DOI  :  10.1186/1471-2180-14-12

 received in 2013-09-04, accepted in 2014-01-16,  发布年份 2014

【 摘 要 】

Background

Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria.

Results

We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg²⁺ at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin.

Conclusions

The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic.

【 授权许可】

   
2014 Meiers et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Laible G, Hakenbeck R: Penicillin-binding proteins in β-lactam-resistant laboratory mutants of Streptococcus pneumoniae. Mol Microbiol 1987, 1:355-363.
• [2]Hakenbeck R, Tornette S, Adkinson NF: Interaction of non-lytic β-lactams with penicillin-binding proteins in Streptococcus pneumoniae. J Gen Microbiol 1987, 133:755-760.
• [3]Hakenbeck R, Martin C, Dowson C, Grebe T: Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants. J Bacteriol 1994, 176:5574-5577.
• [4]Laible G, Hakenbeck R: Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae. J Bacteriol 1991, 173:6986-6990.
• [5]Krauß J, van der Linden M, Grebe T, Hakenbeck R: Penicillin-binding proteins 2x and 2b as primary PBP-targets in Streptococcus pneumoniae. Microb Drug Resist 1996, 2:183-186.
• [6]Hakenbeck R, Grebe T, Zähner D, Stock JB: β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non penicillin-binding proteins. Mol Microbiol 1999, 33:673-678.
• [7]Grebe T, Paik J, Hakenbeck R: A novel resistance mechanism for β-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferases. J Bacteriol 1997, 179:3342-3349.
• [8]Li L, Storm P, Karlsson OP, Berg S, Wieslander A: Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface. Biochemistry 2003, 42:9677-9686.
• [9]Edman M, Berg S, Storm P, Wikström M, Vikström S, Öhmann A, Wieslander A: Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae. J Biol Chem 2003, 278:8420-8428.
• [10]Berg S, Edman M, Li L, Wikström M, Wieslander A: Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea. J Biol Chem 2001, 276:22056-22063.
• [11]Tatituri RV, Brenner MB, Turk J, Hsu FF: Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. J Mass Spectrom 2012, 47:115-123.
• [12]Brundish DE, Shaw N, Baddiley J: The phospholipids of Pneumococcus I-192R, A.T.C.C. 12213. Some structural rearrangements occurring under mild conditions. Biochem J 1967, 104:205-211.
• [13]Wieslander A, Christiansson A, Rilfors L, Lindblom G: Lipid bilayer stability in membranes, Regulation of lipid composition in Acholeplasma laidlawii as governed by molecular shape. Biochemistry 1980, 19:3650-3655.
• [14]Seo HS, Cartee RT, Pritchard DG, Nahm MH: A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model. J Bacteriol 2008, 190:2379-2387.
• [15]Song JH, Ko KS, Lee JY, Baek JY, Oh WS, Yoon HS, Jeong JY, Chun J: Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis. Mol Cells 2005, 19:365-374.
• [16]Laursen BS, Sørensen HP, Mortensen KK, Sperling-Petersen HU: Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev 2005, 69:101-123.
• [17]Denapaite D, Brückner R, Nuhn M, Reichmann P, Henrich B, Maurer P, Schähle Y, Selbmann P, Zimmermann W, Wambutt R, et al.: The genome of Streptococcus mitis B6 - what is a commensal? PLoS ONE 2010, 5:e9426.
• [18]Reichmann P, Nuhn M, Denapaite D, Brückner R, Henrich B, Maurer P, Rieger M, Klages S, Reinhard R, Hakenbeck R: Genome of Streptococcus oralis strain Uo5. J Bacteriol 2011, 193:2888-2889.
• [19]Czyz A, Wegrzyn G: The Obg subfamily of bacterial GTP-binding proteins: essential proteins of largely unknown functions that are evolutionarily conserved from bacteria to humans. Acta Biochim Pol 2005, 52:35-43.
• [20]Hoskins J, Alborn WEJ, Arnold J, Blaszczak LC, Burgett S, DeHoff BS, Estrem ST, Fritz L, Fu D-J, Fuller W, et al.: Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 2001, 183:5709-5717.
• [21]Sauerbier J, Maurer P, Rieger M, Hakenbeck R: Streptococcus pneumoniae R6 interspecies transformation: genetic analysis of penicillin resistance determinants and genome-wide recombination events. Mol Microbiol 2012, 86:692-706.
• [22]Fani F, Brotherton MC, Leprohon P, Ouellette M: Genomic analysis and reconstruction of cefotaxime resistance in Streptococcus pneumoniae. J Antimicrob Chemother 2013, 68:1718-1727.
• [23]Shaw N: Bacterial glycolipids. Bacteriol Rev 1970, 34:365-377.
• [24]Rottem S: Transbilayer distribution of lipids in microbial membranes. Curr Top Membr Trans 1982, 17:235-261.
• [25]Weik M, Patzelt H, Zaccai G, Oesterhelt D: Localization of glycolipids in membranes by in vivo labeling and neutron diffraction. Mol Cell 1998, 1:411-419.
• [26]Henderson R, Jubb JS, Whytock S: Specific labelling of the protein and lipid on the extracellular surface of purple membrane. J Mol Biol 1978, 123:259-274.
• [27]Kamio Y, Nikaido H: Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium. Biochemistry 1976, 15:2561-2570.
• [28]Campelo F, McMahon HT, Kozlov MM: The hydrophobic insertion mechanism of membrane curvature generation by proteins. Biophys J 2008, 95:2325-2339.
• [29]Wikström M, Kelly AA, Georgiev A, Eriksson HM, Klement MR, Bogdanov M, Dowhan W, Wieslander A: Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function. J Biol Chem 2009, 284:954-965.
• [30]Lopez CS, Alice AF, Heras H, Rivas EA, Sanchez-Rivas C: Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity. Microbiology 2006, 152:605-616.
• [31]Becker P, Hakenbeck R, Henrich B: An ABC transporter of Streptococcus pneumoniae involved in susceptibility to vancoresmycin and bacitracin. Antimicrob Agents Chemother 2009, 53:2034-2041.
• [32]Fischer W: Lipoteichoic acid and lipoglycans. In Bacterial Cell Wall. Edited by Ghuysen J-M, Hakenbeck R. Amsterdam: Elsevier Sciences BV; 1994:199-211.
• [33]Rahman O, Dover LG, Sutcliffe IC: Lipoteichoic acid biosynthesis: two steps forwards, one step sideways? Trends Microbiol 2009, 17:219-225.
• [34]Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas B, Henseler K, Götz F, Zähringer U: A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity. Mol Microbiol 2007, 65:1078-1091.
• [35]Kiriukhin MY, Debabov DV, Shinabarger DL, Neuhaus FC: Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase. J Bacteriol 2001, 183:3506-3514.
• [36]Jorasch P, Wolter FP, Zähringer U, Heinz E: A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products. Mol Microbiol 1998, 29:419-430.
• [37]Webb AJ, Karatsa-Dodgson M, Grundling A: Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes. Mol Microbiol 2009, 74:299-314.
• [38]Doran KS, Engelson EJ, Khosravi A, Maisey HC, Fedtke I, Equils O, Michelsen KS, Arditi M, Peschel A, Nizet V: Blood–brain barrier invasion by group B Streptococcus depends upon proper cell-surface anchoring of lipoteichoic acid. J Clin Invest 2005, 115:2499-2507.
• [39]Theilacker C, Sanchez-Carballo P, Toma I, Fabretti F, Sava I, Kropec A, Holst O, Huebner J: Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis. Mol Microbiol 2009, 71:1055-1069.
• [40]Pakkiri LS, Wolucka BA, Lubert EJ, Waechter CJ: Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus. Glycobiology 2004, 14:73-81.
• [41]Pakkiri LS, Waechter CJ: Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus. Glycobiology 2005, 15:291-302.
• [42]Fischer W: Pneumococcal lipoteichoic and teichoic acid. In Streptococcus pneumoniae - Molecular biology and mechanism of disease. Edited by Tomasz A. Larchmont, NY: Mary Ann Liebert, Inc; 2000:155-177. 10538
• [43]Denapaite D, Brückner R, Hakenbeck R, Vollmer W: Biosynthesis of teichoic acids in Streptococcus pneumoniae and closely related species: lessons from genomes. Microb Drug Resist 2012, 18:344-358.
• [44]Hakenbeck R, Madhour A, Denapaite D, Brückner R: Versatility of choline metabolism and choline binding proteins in Streptococcus pneumoniae and commensal streptococci. FEMS Microbiol Rev 2009, 33:572-586.
• [45]Lacks S, Hotchkiss RD: A study of the genetic material determining an enzyme activity in pneumococcus. Biochim Biophys Acta 1960, 39:508-517.
• [46]Alloing G, Granadel C, Morrison DA, Claverys J-P: Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae. Mol Microbiol 1996, 21:471-478.
• [47]Mascher T, Merai M, Balmelle N, de Saizieu A, Hakenbeck R: The Streptococcus pneumoniae cia regulon: CiaR target sites and transcription profile analysis. J Bacteriol 2003, 185:60-70.
• [48]Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Plainview, New York: Cold Spring Harbor Laboratory Press; 1989.
• [49]Kovács M, Halfmann A, Fedtke I, Heintz M, Peschel A, Vollmer W, Hakenbeck R, Brückner R: A functional dlt operon, encoding proteins required for incorporation of D-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae. J Bacteriol 2006, 188:5797-5805.
• [50]Sung CK, Li H, Claverys JP, Morrison DA: An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 2001, 67:5190-5196.
• [51]Salles C, Creancier L, Claverys JP, Méjean V: The high level streptomycin resistance gene from Streptococcus pneumoniae is a homologue of the ribosomal protein S12 gene from Escherichia coli. Nucleic Acids Res 1992, 20:6103.
• [52]Halfmann A, Hakenbeck R, Brückner R: A new integrative reporter plasmid for Streptococcus pneumoniae. FEMS Microbiol Lett 2007, 268:217-224.
• [53]Arbogast LY, Henderson TO: Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum. J Bacteriol 1975, 123:962-971.
• [54]Hakenbeck R, Ellerbrok H, Briese T, Handwerger S, Tomasz A: Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the β-lactam binding site. Antimicrob Agents Chemother 1986, 30:553-558.
• [55]McKessar S, Hakenbeck R: The two-component regulatory system TCS08 is involved in cellobiose metabolism of Streptococcus pneumoniae R6. J Bacteriol 2007, 189:1342-1350.
• [56]Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ, et al.: Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001, 293:498-506.
• [57]Ottolenghi E, Hotchkiss RD: Release of genetic transforming agent from pneumococcal cultures during growth and disintegration. J Exp Med 1962, 116:491-519.