Inactivation of the dlt Operon inStaphylococcus aureus Confers Sensitivity to Defensins, Protegrins, and Other Antimicrobial Peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms.Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked d-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1–3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with thedlt genes involved in the transfer of d-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of d-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of thed-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems. Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms.Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked d-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1–3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with thedlt genes involved in the transfer of d-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of d-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of thed-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems. Antimicrobial peptides play an important role in the defense of insects, vertebrates, and humans against pathogenic microorganisms (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar) and have accordingly been designated “host defense peptides.” Substances such as defensins from the granules of phagocytes, epithelial surfaces, and skin (2Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1994; 6: 584-589Crossref PubMed Scopus (378) Google Scholar), protegrins from porcine leukocytes (3Kokryakov V.N. Harwig S.S.L. Panyutich E.A. Shevchenko A.A. Aleshina G.M. Shamova O.V. Korneva H.A. Lehrer R.I. FEBS Lett. 1993; 327: 231-236Crossref PubMed Scopus (433) Google Scholar), tachyplesins from the hemocytes of the horseshoe crab (4Iwanaga S. Muta T. Shigenaga T. Seki N. Kawano K. Katsu T. Kawabata S. CIBA Found. Symp. 1994; 186: 160-175PubMed Google Scholar), and magainins from amphibian skin (5Berkowitz B.A. Bevins C.L. Zasloff M.A. Biochem. Pharmacol. 1990; 39: 625-629Crossref PubMed Scopus (74) Google Scholar) share an amphiphilic cationic structure and a membrane-damaging activity by forming pores or disintegrating the cytoplasmic membrane bilayer. Peptides with similar structure and activity are also produced by many Gram-positive bacteria and include the nonribosomally synthesized gramicidins and polymyxin B (6Kleinkauf H. von Döhren H. Eur. J. Biochem. 1990; 192: 1-15Crossref PubMed Scopus (239) Google Scholar), ribosomally synthesized peptides, such as lactococcin A or pediocin PA-1 (7Nes I.F. Diep D.B. Havarstein L.S. Brurberg M.B. Eijsink V. Holo H. Antonie Leeuwenhoek. 1996; 70: 113-128Crossref PubMed Scopus (629) Google Scholar), and lantibiotics, which contain the posttranslationally formed thioether amino acid lanthionine (8Sahl H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar). Because of their unique structural features and biotechnological importance, lantibiotics such as nisin, subtilin, Pep 5, epidermin, and gallidermin have been extensively investigated (8Sahl H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar, 9Peschel A. Schnell N. Hille M. Entian K.-D. Götz F. Mol. Gen. Genet. 1997; 254: 312-318Crossref PubMed Scopus (45) Google Scholar).Staphylococcus aureus, a major human pathogen, tolerates high concentrations of several host defense peptides and lantibiotics. Strains resistant to defensin-like platelet microbicidal proteins have been shown to be more virulent than sensitive ones (10Wu T. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1994; 38: 729-732Crossref PubMed Scopus (65) Google Scholar). The mechanisms responsible for the resistance phenotype are unknown. The broad range of antimicrobial peptides tolerated distinguishes them from the highly specific self-protection systems of lantibiotic-producing staphylococcal strains (11Peschel A. Götz F. J. Bacteriol. 1996; 178: 531-536Crossref PubMed Google Scholar, 12Otto M. Peschel A. Götz F. FEMS Microb. Lett. 1998; 166: 203-211PubMed Google Scholar).Gram-positive bacteria are not protected by an outer membrane. TheS. aureus cell wall is instead formed by a thick peptidoglycan fabric and by polymers of alternating phosphate and alditol groups called teichoic acids. These polymer chains are either covalently connected to the peptidoglycan (wall teichoic acids, WTA) 1The abbreviations used are:WTA, wall teichoic acid(s); LTA, lipoteichoic acid(s); PCR, polymerase chain reaction; RP-HPLC, reversed-phase high-performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s).1The abbreviations used are:WTA, wall teichoic acid(s); LTA, lipoteichoic acid(s); PCR, polymerase chain reaction; RP-HPLC, reversed-phase high-performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s). (13Pooley H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers B. V., Amsterdam, The Netherlands1994Google Scholar) or to membrane glycolipids (lipoteichoic acids, LTA) (14Fischer W. Adv. Microbiol. Physiol. 1988; 29: 233-302Crossref PubMed Scopus (221) Google Scholar). Teichoic acids of the various Gram-positive species are highly variable in the use of alditol groups (glycerol or ribitol) and in modifications of the alditol with glycosyl residues or d-alanine. The highly charged teichoic acids are essential for viability and seem to be involved in the control of cell shape, autolytic enzymes, and magnesium ion concentration within the cell envelope (13Pooley H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers B. V., Amsterdam, The Netherlands1994Google Scholar, 15Bierbaum G. Sahl H.-G. J. Bacteriol. 1987; 169: 5452-5458Crossref PubMed Google Scholar).In an attempt to understand the mechanisms by which staphylococci resist antimicrobial peptides, we isolated gallidermin-sensitiveStaphylococcus xylosus and Staphylococcus aureusmutants, analysis of which revealed that the absence ofd-alanine esters from teichoic acids leads to increased sensitivity toward cationic antimicrobial peptides.DISCUSSIONThe dltABCD genes of S. aureus, S. xylosus, L. casei, and B. subtilis are similar in sequence and organization. Studies in L. caseihave demonstrated a role of DltA as ad-alanine-d-alanyl carrier protein ligase (Dcl), which activates d-alanine by hydrolysis of ATP and transfers it to the phosphopantetheine cofactor of a specificd-alanine carrier protein (Dcp), which is encoded bydltC (33Debabov D.V. Heaton M.P. Zhang Q. Stewart K.D. Lambalot R.H. Neuhaus F.C. J. Bacteriol. 1996; 178: 2869-3876Crossref Google Scholar, 34Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar) (Fig. 4). The hydrophobic DltB is indispensable for d-alanine incorporation into teichoic acids and may be involved in the transfer of activated d-alanine across the cytoplasmic membrane (35Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) (Fig. 4). The essential role of DltD and the presence of a putative N-terminal signal peptide suggest an involvement in the transfer ofd-alanine from the membrane carrier to teichoic acids (Fig.4).The increased sensitivity of S. aureus and S. xylosus dlt mutants toward a variety of membrane-active antimicrobial peptides provides evidence for a role of the d-alanine substituents in the protection of the bacteria against these substances. Inactivation of dlt genes caused considerably lower minimal inhibitory concentrations of (i) host defense peptides with β-sheet structure and disulfide bridges, such as human defensin HNP1–3, protegrins from porcine leukocytes, and tachyplesins from horseshoe crab hemocytes; (ii) the linear peptide magainin II from amphibian skin; and (iii) the bacteria-derived lantibiotics gallidermin and nisin, which contain thioether bridges. Since the common structural feature of these molecules is a positive net charge, and since the sensitivity toward neutral gramicidin D was the same in the wild-type and the dlt mutants, we propose that the basis for increased sensitivity is an altered electrostatic interaction of the peptides with the mutant cells. The teichoic acid backbone is highly charged by deprotonized phosphate groups, and esterification withd-alanine causes a reduction of the net negative charge by introduction of basic amino groups (Fig. 4). Accordingly, the S. aureus and S. xylosus dlt mutants, whose teichoic acids are devoid of d-alanine, bound lower amounts of negatively charged green fluorescent protein but higher amounts of positively charged cytochrome c and gallidermin, while strains with increased d-alanine content showed the opposite behavior. Increased accumulation of antimicrobial peptides in the vicinity of the cytoplasmic membrane is therefore very likely to be the basis for the higher sensitivity of the dlt mutants.Cationic properties are necessary for the initial interaction of membrane-damaging peptides with the negatively charged membrane surface (38Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Reduction of the negative cell envelope charge by incorporation of d-alanine may thus be regarded as a multiple drug protection mechanism. A similar observation has been made with Gram-negative bacteria whose resistance to the cationic peptides polymyxin B and protamine was caused by a reduction of the anionic nature of the lipopolysaccharide (39Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (171) Google Scholar). Bacteria have to cope with antimicrobial peptides in many environments. The production of bacteriocin-like molecules such as gallidermin or nisin is a prevalent strategy among microorganisms to inhibit the growth of competing strains (7Nes I.F. Diep D.B. Havarstein L.S. Brurberg M.B. Eijsink V. Holo H. Antonie Leeuwenhoek. 1996; 70: 113-128Crossref PubMed Scopus (629) Google Scholar, H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar) in higher to the innate immunity against bacterial infections (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar). bacteria such as which in with humans and mechanisms against host defense peptides are of Accordingly, a the of S. aureus, and to and the in resistance to defensin-like platelet microbicidal proteins has been demonstrated (10Wu T. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1994; 38: 729-732Crossref PubMed Scopus (65) Google Scholar). the mechanisms by which bacteria are to the human defense systems has a the of infections and the for antimicrobial the the resistance to host defense peptides in a Gram-positive teichoic acids occur in many Gram-positive human and (14Fischer W. Adv. Microbiol. Physiol. 1988; 29: 233-302Crossref PubMed Scopus (221) Google Scholar), and the the d-alanine esters or resistance systems against host defense peptides to the and of these bacteria. Antimicrobial peptides play an important role in the defense of insects, vertebrates, and humans against pathogenic microorganisms (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar) and have accordingly been designated “host defense peptides.” Substances such as defensins from the granules of phagocytes, epithelial surfaces, and skin (2Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1994; 6: 584-589Crossref PubMed Scopus (378) Google Scholar), protegrins from porcine leukocytes (3Kokryakov V.N. Harwig S.S.L. Panyutich E.A. Shevchenko A.A. Aleshina G.M. Shamova O.V. Korneva H.A. Lehrer R.I. FEBS Lett. 1993; 327: 231-236Crossref PubMed Scopus (433) Google Scholar), tachyplesins from the hemocytes of the horseshoe crab (4Iwanaga S. Muta T. Shigenaga T. Seki N. Kawano K. Katsu T. Kawabata S. CIBA Found. Symp. 1994; 186: 160-175PubMed Google Scholar), and magainins from amphibian skin (5Berkowitz B.A. Bevins C.L. Zasloff M.A. Biochem. Pharmacol. 1990; 39: 625-629Crossref PubMed Scopus (74) Google Scholar) share an amphiphilic cationic structure and a membrane-damaging activity by forming pores or disintegrating the cytoplasmic membrane bilayer. Peptides with similar structure and activity are also produced by many Gram-positive bacteria and include the nonribosomally synthesized gramicidins and polymyxin B (6Kleinkauf H. von Döhren H. Eur. J. Biochem. 1990; 192: 1-15Crossref PubMed Scopus (239) Google Scholar), ribosomally synthesized peptides, such as lactococcin A or pediocin PA-1 (7Nes I.F. Diep D.B. Havarstein L.S. Brurberg M.B. Eijsink V. Holo H. Antonie Leeuwenhoek. 1996; 70: 113-128Crossref PubMed Scopus (629) Google Scholar), and lantibiotics, which contain the posttranslationally formed thioether amino acid lanthionine (8Sahl H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar). Because of their unique structural features and biotechnological importance, lantibiotics such as nisin, subtilin, Pep 5, epidermin, and gallidermin have been extensively investigated (8Sahl H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar, 9Peschel A. Schnell N. Hille M. Entian K.-D. Götz F. Mol. Gen. Genet. 1997; 254: 312-318Crossref PubMed Scopus (45) Google Scholar). Staphylococcus aureus, a major human pathogen, tolerates high concentrations of several host defense peptides and lantibiotics. Strains resistant to defensin-like platelet microbicidal proteins have been shown to be more virulent than sensitive ones (10Wu T. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1994; 38: 729-732Crossref PubMed Scopus (65) Google Scholar). The mechanisms responsible for the resistance phenotype are unknown. The broad range of antimicrobial peptides tolerated distinguishes them from the highly specific self-protection systems of lantibiotic-producing staphylococcal strains (11Peschel A. Götz F. J. Bacteriol. 1996; 178: 531-536Crossref PubMed Google Scholar, 12Otto M. Peschel A. Götz F. FEMS Microb. Lett. 1998; 166: 203-211PubMed Google Scholar). Gram-positive bacteria are not protected by an outer membrane. TheS. aureus cell wall is instead formed by a thick peptidoglycan fabric and by polymers of alternating phosphate and alditol groups called teichoic acids. These polymer chains are either covalently connected to the peptidoglycan (wall teichoic acids, WTA) 1The abbreviations used are:WTA, wall teichoic acid(s); LTA, lipoteichoic acid(s); PCR, polymerase chain reaction; RP-HPLC, reversed-phase high-performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s).1The abbreviations used are:WTA, wall teichoic acid(s); LTA, lipoteichoic acid(s); PCR, polymerase chain reaction; RP-HPLC, reversed-phase high-performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s). (13Pooley H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers B. V., Amsterdam, The Netherlands1994Google Scholar) or to membrane glycolipids (lipoteichoic acids, LTA) (14Fischer W. Adv. Microbiol. Physiol. 1988; 29: 233-302Crossref PubMed Scopus (221) Google Scholar). Teichoic acids of the various Gram-positive species are highly variable in the use of alditol groups (glycerol or ribitol) and in modifications of the alditol with glycosyl residues or d-alanine. The highly charged teichoic acids are essential for viability and seem to be involved in the control of cell shape, autolytic enzymes, and magnesium ion concentration within the cell envelope (13Pooley H.M. Karamata D. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers B. V., Amsterdam, The Netherlands1994Google Scholar, 15Bierbaum G. Sahl H.-G. J. Bacteriol. 1987; 169: 5452-5458Crossref PubMed Google Scholar). an attempt to understand the mechanisms by which staphylococci resist antimicrobial peptides, we isolated gallidermin-sensitiveStaphylococcus xylosus and Staphylococcus aureusmutants, analysis of which revealed that the absence ofd-alanine esters from teichoic acids leads to increased sensitivity toward cationic antimicrobial peptides. dltABCD genes of S. aureus, S. xylosus, L. casei, and B. subtilis are similar in sequence and organization. Studies in L. caseihave demonstrated a role of DltA as ad-alanine-d-alanyl carrier protein ligase (Dcl), which activates d-alanine by hydrolysis of ATP and transfers it to the phosphopantetheine cofactor of a specificd-alanine carrier protein (Dcp), which is encoded bydltC (33Debabov D.V. Heaton M.P. Zhang Q. Stewart K.D. Lambalot R.H. Neuhaus F.C. J. Bacteriol. 1996; 178: 2869-3876Crossref Google Scholar, 34Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar) (Fig. 4). The hydrophobic DltB is indispensable for d-alanine incorporation into teichoic acids and may be involved in the transfer of activated d-alanine across the cytoplasmic membrane (35Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) (Fig. 4). The essential role of DltD and the presence of a putative N-terminal signal peptide suggest an involvement in the transfer ofd-alanine from the membrane carrier to teichoic acids (Fig.4).The increased sensitivity of S. aureus and S. xylosus dlt mutants toward a variety of membrane-active antimicrobial peptides provides evidence for a role of the d-alanine substituents in the protection of the bacteria against these substances. Inactivation of dlt genes caused considerably lower minimal inhibitory concentrations of (i) host defense peptides with β-sheet structure and disulfide bridges, such as human defensin HNP1–3, protegrins from porcine leukocytes, and tachyplesins from horseshoe crab hemocytes; (ii) the linear peptide magainin II from amphibian skin; and (iii) the bacteria-derived lantibiotics gallidermin and nisin, which contain thioether bridges. Since the common structural feature of these molecules is a positive net charge, and since the sensitivity toward neutral gramicidin D was the same in the wild-type and the dlt mutants, we propose that the basis for increased sensitivity is an altered electrostatic interaction of the peptides with the mutant cells. The teichoic acid backbone is highly charged by deprotonized phosphate groups, and esterification withd-alanine causes a reduction of the net negative charge by introduction of basic amino groups (Fig. 4). Accordingly, the S. aureus and S. xylosus dlt mutants, whose teichoic acids are devoid of d-alanine, bound lower amounts of negatively charged green fluorescent protein but higher amounts of positively charged cytochrome c and gallidermin, while strains with increased d-alanine content showed the opposite behavior. Increased accumulation of antimicrobial peptides in the vicinity of the cytoplasmic membrane is therefore very likely to be the basis for the higher sensitivity of the dlt mutants.Cationic properties are necessary for the initial interaction of membrane-damaging peptides with the negatively charged membrane surface (38Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Reduction of the negative cell envelope charge by incorporation of d-alanine may thus be regarded as a multiple drug protection mechanism. A similar observation has been made with Gram-negative bacteria whose resistance to the cationic peptides polymyxin B and protamine was caused by a reduction of the anionic nature of the lipopolysaccharide (39Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (171) Google Scholar). Bacteria have to cope with antimicrobial peptides in many environments. The production of bacteriocin-like molecules such as gallidermin or nisin is a prevalent strategy among microorganisms to inhibit the growth of competing strains (7Nes I.F. Diep D.B. Havarstein L.S. Brurberg M.B. Eijsink V. Holo H. Antonie Leeuwenhoek. 1996; 70: 113-128Crossref PubMed Scopus (629) Google Scholar, H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar) in higher to the innate immunity against bacterial infections (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar). bacteria such as which in with humans and mechanisms against host defense peptides are of Accordingly, a the of S. aureus, and to and the in resistance to defensin-like platelet microbicidal proteins has been demonstrated (10Wu T. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1994; 38: 729-732Crossref PubMed Scopus (65) Google Scholar). the mechanisms by which bacteria are to the human defense systems has a the of infections and the for antimicrobial the the resistance to host defense peptides in a Gram-positive teichoic acids occur in many Gram-positive human and (14Fischer W. Adv. Microbiol. Physiol. 1988; 29: 233-302Crossref PubMed Scopus (221) Google Scholar), and the the d-alanine esters or resistance systems against host defense peptides to the and of these bacteria. The dltABCD genes of S. aureus, S. xylosus, L. casei, and B. subtilis are similar in sequence and organization. Studies in L. caseihave demonstrated a role of DltA as ad-alanine-d-alanyl carrier protein ligase (Dcl), which activates d-alanine by hydrolysis of ATP and transfers it to the phosphopantetheine cofactor of a specificd-alanine carrier protein (Dcp), which is encoded bydltC (33Debabov D.V. Heaton M.P. Zhang Q. Stewart K.D. Lambalot R.H. Neuhaus F.C. J. Bacteriol. 1996; 178: 2869-3876Crossref Google Scholar, 34Heaton M.P. Neuhaus F.C. J. Bacteriol. 1992; 174: 4707-4717Crossref PubMed Google Scholar) (Fig. 4). The hydrophobic DltB is indispensable for d-alanine incorporation into teichoic acids and may be involved in the transfer of activated d-alanine across the cytoplasmic membrane (35Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) (Fig. 4). The essential role of DltD and the presence of a putative N-terminal signal peptide suggest an involvement in the transfer ofd-alanine from the membrane carrier to teichoic acids The increased sensitivity of S. aureus and S. xylosus dlt mutants toward a variety of membrane-active antimicrobial peptides provides evidence for a role of the d-alanine substituents in the protection of the bacteria against these substances. Inactivation of dlt genes caused considerably lower minimal inhibitory concentrations of (i) host defense peptides with β-sheet structure and disulfide bridges, such as human defensin HNP1–3, protegrins from porcine leukocytes, and tachyplesins from horseshoe crab hemocytes; (ii) the linear peptide magainin II from amphibian skin; and (iii) the bacteria-derived lantibiotics gallidermin and nisin, which contain thioether bridges. Since the common structural feature of these molecules is a positive net charge, and since the sensitivity toward neutral gramicidin D was the same in the wild-type and the dlt mutants, we propose that the basis for increased sensitivity is an altered electrostatic interaction of the peptides with the mutant cells. The teichoic acid backbone is highly charged by deprotonized phosphate groups, and esterification withd-alanine causes a reduction of the net negative charge by introduction of basic amino groups (Fig. 4). Accordingly, the S. aureus and S. xylosus dlt mutants, whose teichoic acids are devoid of d-alanine, bound lower amounts of negatively charged green fluorescent protein but higher amounts of positively charged cytochrome c and gallidermin, while strains with increased d-alanine content showed the opposite behavior. Increased accumulation of antimicrobial peptides in the vicinity of the cytoplasmic membrane is therefore very likely to be the basis for the higher sensitivity of the dlt properties are necessary for the initial interaction of membrane-damaging peptides with the negatively charged membrane surface (38Shai Y. Trends Biochem. Sci. 1995; 20: 460-464Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Reduction of the negative cell envelope charge by incorporation of d-alanine may thus be regarded as a multiple drug protection mechanism. A similar observation has been made with Gram-negative bacteria whose resistance to the cationic peptides polymyxin B and protamine was caused by a reduction of the anionic nature of the lipopolysaccharide (39Nummila K. Kilpeläinen I. Zähringer U. Vaara M. Helander I.M. Mol. Microbiol. 1995; 16: 271-278Crossref PubMed Scopus (171) Google Scholar). Bacteria have to cope with antimicrobial peptides in many environments. The production of bacteriocin-like molecules such as gallidermin or nisin is a prevalent strategy among microorganisms to inhibit the growth of competing strains (7Nes I.F. Diep D.B. Havarstein L.S. Brurberg M.B. Eijsink V. Holo H. Antonie Leeuwenhoek. 1996; 70: 113-128Crossref PubMed Scopus (629) Google Scholar, H.-G. Jack R.W. Bierbaum G. Eur. J. Biochem. 1995; 230: 827-853Crossref PubMed Scopus (257) Google Scholar) in higher to the innate immunity against bacterial infections (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar). bacteria such as which in with humans and mechanisms against host defense peptides are of Accordingly, a the of S. aureus, and to and the in resistance to defensin-like platelet microbicidal proteins has been demonstrated (10Wu T. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1994; 38: 729-732Crossref PubMed Scopus (65) Google Scholar). the mechanisms by which bacteria are to the human defense systems has a the of infections and the for antimicrobial the the resistance to host defense peptides in a Gram-positive teichoic acids occur in many Gram-positive human and (14Fischer W. Adv. Microbiol. Physiol. 1988; 29: 233-302Crossref PubMed Scopus (221) Google Scholar), and the the d-alanine esters or resistance systems against host defense peptides to the and of these bacteria. We for for mutants, and for human