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A Novel Secondary Acyl Chain in the Lipopolysaccharide of Bordetella pertussis Required for Efficient Infection of Human Macrophages

2007; Elsevier BV; Volume: 282; Issue: 52 Linguagem: Inglês

10.1074/jbc.m706391200

ISSN

1083-351X

Autores

Jeroen Geurtsen, Eline Angevaare, Marlieke Janssen, Hendrik-Jan Hamstra, Jan ten Hove, Alex de Haan, Betsy Kuipers, Jan Tommassen, Peter van der Ley,

Tópico(s)

Salmonella and Campylobacter epidemiology

Resumo

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2′ and 3′ positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2′ position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages. Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2′ and 3′ positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2′ position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages. Pertussis or whooping cough is a severe acute respiratory illness that is characterized by paroxysmal coughing and a distinctive "whooping" sound when air is subsequently inhaled. The disease is highly contagious and most severe in neonates and children younger than one year. Pertussis is caused by the Gram-negative bacterium Bordetella pertussis. While the genus Bordetella currently encompasses nine species, apart from B. pertussis only three other members, Bordetella bronchiseptica, Bordetella parapertussis, and Bordetella holmesii, have been associated with respiratory infections in humans and other mammals (1Mattoo S. Cherry J.D. Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (842) Google Scholar). The Gram-negative bacterial cell envelope is composed of two membranes, the inner and the outer membrane, which are separated by the periplasm. The inner membrane is a symmetrical bilayer composed of phospholipids, whereas the outer membrane is asymmetric and consists of phospholipids in the inner leaflet and lipopolysaccharide (LPS) 2The abbreviations used are: LPSlipopolysaccharideS. typhimuriumSalmonella enterica serovar TyphimuriumLBLuria Bertani brothBGBordet-GengouMSmass spectrometryMS/MStandem MS3OH C103-hydroxydecanoic acid3OH C123-hydroxydodecanoic acid3OH C143-hydroxytetradecanoic acid2OH C122-hydroxydodecanoic acidC12laurateCFUcolony forming unitMM6MonoMac 6ILinterleukinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 2The abbreviations used are: LPSlipopolysaccharideS. typhimuriumSalmonella enterica serovar TyphimuriumLBLuria Bertani brothBGBordet-GengouMSmass spectrometryMS/MStandem MS3OH C103-hydroxydecanoic acid3OH C123-hydroxydodecanoic acid3OH C143-hydroxytetradecanoic acid2OH C122-hydroxydodecanoic acidC12laurateCFUcolony forming unitMM6MonoMac 6ILinterleukinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine in the outer leaflet. LPS, which is also known as endotoxin, consists of three distinct structural domains: lipid A, the core, and the O-antigen (2Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). The first domain, lipid A, functions as a hydrophobic membrane anchor and forms the bioactive component of the molecule (3Takada H. Kotani S. Crit. Rev. Microbiol. 1989; 16: 477-523Crossref PubMed Scopus (120) Google Scholar). The structure of lipid A is conserved among different bacterial groups, indicating its importance for the correct functioning of the outer membrane. Generally, lipid A consists of a β-1′,6-linked d-glucosamine (GlcN) disaccharide carrying ester- and amide-linked 3-hydroxyl fatty acids at the C-2, C-3, C-2′, and C-3′ positions, and phosphate groups at positions C-1 and C-4′. The endotoxic activity of LPS is based on the recognition of lipid A by the TLR4/MD-2 complex of the host, which leads to the activation of NF-κB and, consequently, to an increased production and secretion of pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-α, and IL-1β (4Pålsson-McDermott E.M. O'Neill L.A.J. Immunology. 2004; 113: 153-162Crossref PubMed Scopus (932) Google Scholar). lipopolysaccharide Salmonella enterica serovar Typhimurium Luria Bertani broth Bordet-Gengou mass spectrometry tandem MS 3-hydroxydecanoic acid 3-hydroxydodecanoic acid 3-hydroxytetradecanoic acid 2-hydroxydodecanoic acid laurate colony forming unit MonoMac 6 interleukin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine lipopolysaccharide Salmonella enterica serovar Typhimurium Luria Bertani broth Bordet-Gengou mass spectrometry tandem MS 3-hydroxydecanoic acid 3-hydroxydodecanoic acid 3-hydroxytetradecanoic acid 2-hydroxydodecanoic acid laurate colony forming unit MonoMac 6 interleukin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Current knowledge about lipid A biosynthesis is mainly derived from studies in Escherichia coli and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), where the biosynthetic pathway has been completely elucidated. It consists of nine enzymes that work in a successive order. In the first step, an acyl chain is transferred from the R-3-hydroxytetradecanoic acid (3OH C14)-acyl carrier protein to the GlcN 3 position of UDP-N-acetyl glucosamine (GlcNAc) by the acyltransferase LpxA (5Crowell D.N. Reznikoff W.S. Raetz C.R.H. J. Bacteriol. 1986; 169: 5727-5734Crossref Google Scholar, 6Coleman J. Raetz C.R.H. J. Bacteriol. 1988; 170: 1268-1274Crossref PubMed Google Scholar). Then, the acylated UDP-GlcNAc is de-acetylated by the LpxC enzyme (7Young K. Silver L.L. Bramhill D. Cameron P. Eveland S.S. Raetz C.R.H. Hyland S.A. Anderson M.S. J. Biol. Chem. 1995; 270: 30384-30391Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), after which LpxD adds a 3-hydroxyl acyl chain at this position (8Kelly T.M. Stachula S.A. Raetz C.R.H. Anderson M.S. J. Biol. Chem. 1993; 268: 19866-19874Abstract Full Text PDF PubMed Google Scholar), resulting in a UDP-2,3-diacylGlcN molecule. Next, UMP is removed from a proportion of the UDP-2,3-diacylGlcN pool by LpxH (9Babinski K.J. Ribeiro A.A. Raetz C.R.H. J. Biol. Chem. 2002; 277: 25937-25946Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), before a tetra-acylated GlcN disaccharide is formed by LpxB (5Crowell D.N. Reznikoff W.S. Raetz C.R.H. J. Bacteriol. 1986; 169: 5727-5734Crossref Google Scholar). After 4′-phosphorylation by LpxK, creating a molecule known as lipid IVA (10Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), two 2-keto-3-deoxyoctulosonic acid residues are added by KdtA (11Clementz T. Raetz C.R.H. J. Biol. Chem. 1991; 266: 9687-9696Abstract Full Text PDF PubMed Google Scholar), and finally the secondary acyl chains are added by the late acyltransferases LpxL and LpxM (12Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 13Clementz T. Zhou Z. Raetz C.R.H. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The late acyltransferase LpxL of E. coli was found to be responsible for the addition of a secondary laurate (C12) moiety to the 2′ position of lipid A (12Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (67) Google Scholar). LpxL homologues have been identified in several other Gram-negative bacteria, including Haemophilus influenzae (15Lee N.G. Sunshine M.G. Engstrom J.J. Gibson B.W. Apicella M.A. J. Biol. Chem. 1995; 270: 27151-27159Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), Neisseria meningitidis (16van der Ley P. Steeghs L. Hamstra H.-J. ten Hove J. Zomer B. van Alphen L. Infect. Immun. 2001; 69: 5981-5990Crossref PubMed Scopus (176) Google Scholar), S. typhimurium (17Sunshine M.G. Gibson B.W. Engstrom J.J. Nichols W.A. Jones B.D. Apicella M.A. J. Bacteriol. 1997; 179: 5521-5533Crossref PubMed Google Scholar), and Yersinia pestis (18Rebeil R. Ernst R.K. Jarrett C.O. Adams K.N. Miller S.I. Hinnebusch B.J. J. Bacteriol. 2006; 188: 1381-1388Crossref PubMed Scopus (72) Google Scholar). The second late acyltransferase, LpxM, is closely related to LpxL and was initially described as a multicopy suppressor of an lpxL mutation (19Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (93) Google Scholar). In E. coli, LpxM is responsible for the addition of a secondary myristate (C14) chain at the 3′ position of lipid A (13Clementz T. Zhou Z. Raetz C.R.H. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Bacteria with mutations in lpxL and lpxM harbor underacylated LPS species, which display a reduced activity in stimulating innate immune responses (16van der Ley P. Steeghs L. Hamstra H.-J. ten Hove J. Zomer B. van Alphen L. Infect. Immun. 2001; 69: 5981-5990Crossref PubMed Scopus (176) Google Scholar, 20Low K.B. Ittensohn M. Le T. Platt J. Sodi S. Amoss M. Ash O. Carmichael E. Chakraborty A. Fischer J. Lin S.L. Luo X. Miller S.I. Zheng L. King I. Pawelek J.M. Bermudes D. Nat. Biotechnol. 1998; 17: 37-41Crossref Scopus (376) Google Scholar, 21Cognet I. de Coignac A.B. Magistrelli G. Jeannin P. Aubry J.P. Maisnier-Patin K. Caron G. Chevalier S. Humbert F. Nguyen T. Beck A. Velin D. Delneste Y. Malissard M. Gauchat J.F. J. Immunol. Methods. 2003; 272: 199-210Crossref PubMed Scopus (36) Google Scholar). The structure of B. pertussis lipid A (Fig. 1) resembles that of E. coli. It typically consists of a GlcN disaccharide substituted with 3OH C14 residues at positions 2, 2′, and 3′ via ester or amide linkage and with an R-3-hydroxydecanoic acid (3OH C10) residue at the 3 position via ester linkage. A secondary C14 replaces the hydroxyl group of 3OH C14 at the 2′ position (Fig. 1) (22Caroff M. Deprun C. Richards J.C. Karibian D. J. Bacteriol. 1994; 176: 5156-5159Crossref PubMed Google Scholar). Limited information on the genetics of Bordetella lipid A biosynthesis is currently available, and detailed analyses have only been performed for the acyl transferase LpxA and the 2-keto-3-deoxyoctulosonic acid transferase KdtA (23Sweet C.R. Preston A. Toland E. Ramirez S.M. Cotter R.J. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 2002; 277: 18281-18290Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 24Isobe T. White K.A. Allen A.G. Peacock M. Raetz C.R.H. Maskell D.J. J. Bacteriol. 1999; 181: 2648-2651Crossref PubMed Google Scholar). The goal of the present study was to identify the gene encoding the enzyme responsible for the attachment of the secondary acyl chain to B. pertussis LPS with the eventual goal to inactivate this gene and create a less reactogenic vaccine strain. We identified a locus of two lpxL homologues in the genome of B. pertussis, which raised the question which of these genes is responsible for the attachment of the single secondary acyl chain and what the function of the other LpxL homologue might be. The study resulted in the identification of new LPS forms in B. pertussis, required for successful infection of human macrophages. Bacterial Strains and Growth Conditions—All bacterial strains used are described in Table 1. Typically, the E. coli strains were grown at 37 °C in a modified Luria-Bertani broth, designated LB (25Tommassen J. van Tol H. Lugtenberg B. EMBO J. 1983; 2: 1275-1279Crossref PubMed Scopus (116) Google Scholar), supplemented with 0.2% glucose or at either 30 °C or 42 °C in a synthetic minimal medium (26Winkler K.C. de Haan P.G. Arch. Biochem. 1948; 18: 97-107PubMed Google Scholar) supplemented with 0.5% glucose, while shaking at 200 rpm. When appropriate, the media were supplemented with 100 μg/ml ampicillin, 10 μg/ml tetracycline, 10 μg/ml gentamicin, 50 μg/ml nalidixic acid, or 300 μg/ml streptomycin, for plasmid maintenance or strain selection. B. pertussis was grown at 35 °C on Bordet-Gengou (BG) agar (Difco) supplemented with 15% defibrinated sheep blood (Biotrading). To induce the expression of the lpxL1 and lpxL2 genes from plasmids in B. pertussis, the bacteria were grown in synthetic THIJS medium (27Thalen M. van den IJssel J. Jiskoot W. Zomer B. Roholl P. de Gooijer C. Beuvery C. Trampen J. J. Biotechnol. 1999; 75: 147-159Crossref PubMed Scopus (76) Google Scholar) supplemented with 1 mm isopropyl-1-thio-β-d-galactopyranoside at 35 °C while shaking at 175 rpm.TABLE 1Bacterial strains and plasmidsStrain or plasmidGenotype or descriptionaStr, streptomycin; Gm, gentamicin; Tet, tetracycline; Amp, ampicillin; Kan, kanamycin.Source or referencebNVI, Netherlands Vaccine Institute, Bilthoven, The Netherlands.B. pertussisB213StrR derivative of B. pertussis strain Tohama(41Kasuga B. Nakase Y. Ukishima K. Takatsu K. Arch. Exp. Med. 1953; 27: 21-28Google Scholar)B213 ΔlpxL1lpxL1 mutant of B213 strain, StrR, GmRThis studyE. coliTOP10F′F′ {lacIq Tn10 (TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) Φ80 lacZΔM15 ΔlacX74 deoR recA 1 araD 139 Δ(ara-leu)7697 galU galK rpsL endA 1 nupGInvitrogenDH5αF−, Δ(lacZYA-algF)U169 thi-1 hsdR17 gyrA96 recA 1 endA 1 supE44 relA 1 phoA Φ80 dlacZΔM15(42Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8098) Google Scholar)SM10λpirthi thr leu fhuA lacY supE recA::RP4-2-Tc::Mu λpir R6K KanRNVIW3110Wild-type strain, F−, λ−NVIMLK53lpxL::Tn10, TetR derivative of W3110(19Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (93) Google Scholar)PlasmidspCRII-TOPOE. coli cloning vector, AmpR KanRInvitrogenpET-11aE. coli high copy expression vector, AmpR, T7 promoterNovagenpMMB67EHBroad host range expression vector, AmpR(43Fürste J.P. Pansegrau W. Frank R. Blocker H. Scholz P. Bagdasarian M. Lanka E. Gene (Amst.). 1986; 48: 119-131Crossref PubMed Scopus (810) Google Scholar)pKAS32Allelic exchange suicide vector, AmpR(44Skorupski K. Taylor R.K. Gene (Amst.). 1996; 169: 47-52Crossref PubMed Scopus (365) Google Scholar)pBSL141E. coli vector harboring gentamicin-resistance cassette, AmpR GmR(45Alexeyev M.F. Shokolenko I.N. Croughan T.P. Gene (Amst.). 1995; 160: 63-67Crossref PubMed Scopus (147) Google Scholar)pLpxL1pET-11a derivative harboring B. pertussis lpxL1This studypLpxL2pET-11a derivative harboring B. pertussis lpxL2This studypMMB67EH-LpxL1pMMB67EH derivative harboring B. pertussis lpxL1This studypMMB67EH-LpxL2pMMB67EH derivative harboring B. pertussis lpxL2This studypCRII-LpxL1uppCRII derivative harboring lpxL1-upstream sequenceThis studypCRII-LpxL1downpCRII derivative harboring lpxL1-downstream sequenceThis studypKAS32-LpxL1KOpKAS32 derivative harboring lpxL1 knockout construct, AmpR, GmRThis studya Str, streptomycin; Gm, gentamicin; Tet, tetracycline; Amp, ampicillin; Kan, kanamycin.b NVI, Netherlands Vaccine Institute, Bilthoven, The Netherlands. Open table in a new tab Recombinant DNA Techniques—All plasmids used are described in Table 1. Plasmid DNA was isolated using the Promega Wizard®Plus Minipreps system. Calf-intestine alkaline phosphatase and restriction endonucleases were used according to the instructions of the manufacturer (Fermentas). DNA fragments were isolated from agarose gels using the Qiagen quick gel extraction kit. Ligations were performed using the rapid DNA ligation kit (Roche Applied Science). The lpxL1 and lpxL2 genes from B. pertussis strain B213 were obtained by PCR. The chromosomal template DNA was prepared by resuspending ∼109 bacteria in 50 μl of distilled water, after which the suspension was heated for 15 min at 95 °C. The suspension was then centrifuged for 1 min at 16,100 × g, after which the supernatant was used as template DNA. The sequences of the forward primers, which contained an NdeI site (underlined), including an ATG start codon, were 5′-AACATATGCTCGTCACCCTGTTA-3′ (lpxL1) and 5′-AACATATGAGCCAATTCAAGA-3′ (lpxL2). The sequences of the reverse primers, which contained a BamHI site (underlined) and included a stop codon, were 5′-AAGGATCCTCATCGTTCGGGTTCCTG-3′ (lpxL1) and 5′-AAGGATCCTCAGTACAGCTTGGGCTT-3′ (lpxL2). The PCRs were performed under the following condition: 50 μl of total reaction volume, 25 pmol of each primer, 0.2 mm dNTPs, 3 μl of template DNA solution, 1.5% dimethyl sulfoxide, and 1.75 units of Expand High Fidelity enzyme mix with buffer supplied by the manufacturer (Roche Applied Science). The temperature program was as follows: 95 °C for 3 min, a cycle of 1 min at 95 °C, 1 min at 60 °C, and 2 min at 72 °C, repeated 30 times, followed by 10 min at 72 °C and subsequent cooling to 4 °C. The PCR products were purified from agarose gel and subsequently cloned into pCRII-TOPO. Plasmid DNA from a correct clone was digested with NdeI and BamHI, and the LpxL-encoding fragments were ligated into NdeI- and BamHI-digested pET-11a. The ligation mixture was used to transform E. coli DH5α using the CaCl2 method (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmids containing the correct inserts were designated pLpxL1 and pLpxL2. The nucleotide sequences of the cloned genes were confirmed by sequencing in both directions. To allow for expression in B. pertussis, the lpxL1 and lpxL2 genes were subcloned into the broad host range, low copy number vector pMMB67EH. To this end, pLpxL1 and pLpxL2 were digested with XbaI and HindIII, and the relevant fragments were ligated into XbaI- and HindIII-digested pMMB67EH. The ligation mixture was used to transform E. coli DH5α. Plasmids with the correct inserts were designated pMMB67EH-LpxL1 and pMMB67EH-LpxL2 (Table 1). The pMMB67EH-based plasmids were used to transform E. coli SM10(λpir), which allowed for subsequent transfer of the plasmids to B. pertussis by conjugation. For complementation experiments, E. coli strains W3110 and MLK53 (Table 1) were transformed with the plasmids. To construct a B. pertussis lpxL1 mutant strain, we amplified a part of the DNA upstream of lpxL1 from B. pertussis strain B213 by using primers 5′-AAATTCGCTCTGGCGCTGCAC-3′ and 5′-AATCAGCACGCGTCTGACCGATGCGAATGAAAGGGCGG-3′, containing an MluI site (underlined). Additionally, a DNA fragment downstream of lpxL1 was obtained by PCR with primers 5′-AAGTCAGACGCGTGCTGAGACAGCGCGCGGCAGGAACC-3′, containing an MluI site (underlined), and 5′-AATCCACGTGATAGCGCCCGGT-3′. Both PCR products were cloned into pCRII-TOPO, resulting in plasmids pCRII-LpxL1up and pCRII-LpxL1down, respectively. An MluI-XbaI fragment of pCRII-LpxL1down was ligated into MluI-XbaI-restricted pCRII-LpxL1up. The resulting plasmid was cut with MluI to allow for insertion of the gentamicin resistance cassette from plasmid pBSL141 obtained by MluI digestion. Finally, an XbaI-SacI fragment of the construct obtained was ligated into the XbaI-SacI-restricted suicide plasmid pKAS32. The final construct, designated pKAS32-LpxL1KO, contained the gentamicin resistance cassette in the reverse orientation relative to the transcription direction of the lpxL1 gene and was used to construct a B. pertussis lpxL1 mutant by allelic exchange. Transformants were screened by PCR using various primer sets. Isolation and Analysis of LPS—LPS was isolated using the hot phenol/water extraction method (29Westphal O. Jann J.K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar) with slight modifications (30Geurtsen J. Steeghs L. Hamstra H.-J. ten Hove J. de Haan A. Kuipers B. Tommassen J. van der Ley P. Infect. Immun. 2006; 74: 5574-5585Crossref PubMed Scopus (60) Google Scholar). The fatty acid composition was analyzed using a 6890 Agilent gas chromatograph (31Welch D.F. Clin. Microbiol. Rev. 1991; 4: 422-438Crossref PubMed Scopus (186) Google Scholar). The lipid A moiety of LPS was isolated as described (30Geurtsen J. Steeghs L. Hamstra H.-J. ten Hove J. de Haan A. Kuipers B. Tommassen J. van der Ley P. Infect. Immun. 2006; 74: 5574-5585Crossref PubMed Scopus (60) Google Scholar) and used for structural analysis by nanoelectrospray tandem mass spectrometry (MS/MS) on a Finnigan LCQ in the negative (MS) or positive (MS/MS) ion mode (32Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1678) Google Scholar). Stimulation of Macrophages and IL-6 Quantification—The human macrophage cell line MonoMac 6 (MM6) (33Ziegler-Heitbrock H.W. Thiel E. Futterer A. Herzog V. Wirtz A. Riethmüller G. Int. J. Cancer. 1988; 41: 456-461Crossref PubMed Scopus (489) Google Scholar) was stimulated with serial dilutions of whole bacterial cell suspensions or purified LPS as described (30Geurtsen J. Steeghs L. Hamstra H.-J. ten Hove J. de Haan A. Kuipers B. Tommassen J. van der Ley P. Infect. Immun. 2006; 74: 5574-5585Crossref PubMed Scopus (60) Google Scholar). The bacterial cell suspensions were prepared by collecting the cells from cultures by centrifugation, after which they were resuspended in phosphate-buffered saline at an optical density at 590 nm (A590) of 1.0, heat-inactivated for 10 min in the presence of 8 mm formaldehyde, and stored at 4 °C. Following stimulation, IL-6 concentrations in the culture supernatants were quantified with an enzyme-linked immunosorbent assay against human IL-6 according to the manufacturer's instructions (PeliKine Compact™). Infection of Human Macrophages—For infection of human macrophages, bacteria were grown for 16 h on fresh BG blood agar plates, after which they were washed once with phosphate-buffered saline and resuspended in 1 ml of Iscove's modified Dulbecco's medium (Invitrogen). Bacteria were added to 5 × 105 MM6 cells, which were maintained in 0.25 ml of pre-warmed Iscove's modified Dulbecco's medium in 24-well tissue culture plates, at a multiplicity of infection of 10 (final volume = 500 μl). After 2 h of incubation (5% CO2 and 37 °C), 100 μg/ml colistin sulfate (end concentration) was added to the wells, after which the plates were incubated further for 2 h at 37°C. Then, the MM6 cells were collected by centrifugation and washed twice with Iscove's modified Dulbecco's medium, after which they were lysed (1 min at 22 °C) in 0.15 ml of phosphate-buffered saline containing 0.1% Triton X-100. The lysed cells were plated onto BG blood agar plates, and the number of viable intracellular bacteria was estimated by determining the number of colony forming units (CFUs) after 72 h of growth. Infection experiments were repeated three times. The colistin sensitivity of the wild-type and mutant strain was determined by growing them in the presence of various concentrations of colistin and, after diluting the suspensions and plating them on BG blood agar plates, counting the number of CFUs. Association of B. pertussis to Human Macrophages—Association of bacteria to MM6 cells was evaluated by flow cytometry. 2 × 109 bacteria were labeled by incubation with 50 μg of fluorescein isothiocyanate in 1 ml of 50 mm carbonate/bicarbonate buffer (pH 9.6) at room temperature for 20 min. The fluorescein isothiocyanate-labeled bacteria were then washed twice with TSA (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm CaCl2, 2mm MgCl2, 0.5% human serum albumin (w/v) (Sigma)), passaged through a 5-μm filter (Millipore) for declumping, and resuspended in TSA to an absorbance at 600 nm of 1. Association to macrophages was assessed in TSA buffer by incubating (45 min at 37 °C or 4 °C) the fluorescein isothiocyanate-labeled bacteria with the macrophages (multiplicity of infection 2, 10, and 50) and, after washing the cells, measuring the percentage of fluorescently labeled macrophages in FL1 by flow cytometry (FACScan, BD Biosciences). Statistical Analysis—Data were statistically analyzed using two-way analysis of variance followed by Bonferroni's multiple comparison test (GraphPad). Alternatively, a Student's t test (two-tailed, two-sample unequal variance) was used. Differences were considered to be significant when p < 0.05. Identification of Late Lipid A Acyltransferase Homologues in B. pertussis—The 306- and 323-amino acid residue sequences of the E. coli K-12 LpxL and LpxM proteins with GenBank™ accession numbers NP_415572 and NP_416369, respectively, were used to identify putative lpxL and lpxM homologues in the complete B. pertussis genome sequence present in the NCBI data base (www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). BLAST search (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar) revealed the presence of two homologues of lpxL and lpxM, i.e. BP3072 and BP3073 with GenBank™ accession numbers NP_881643 and NP_881644, respectively. BP3072 and BP3073 show sequence identities of 21 and 29% to E. coli LpxM, respectively, of 23 and 31% to E. coli LpxL, respectively, and of 25% to each other. Because both proteins show a higher sequence identity to E. coli LpxL than to E. coli LpxM, BP3072 and BP3073 were designated lpxL1 and lpxL2, respectively. The open reading frames are adjacent to one another, with the stop codon of lpxL1 overlapping with the start codon of lpxL2, and, therefore, seem to form an operon. Upstream, in the reverse orientation, and downstream of the operon, genes are located putatively encoding a homologue of the S-adenosyl-methionine synthetase MetK and of the diaminopimelate epimerase DapF, respectively. Further BLAST analysis revealed the presence of lpxL1 and lpxL2 homologues in B. parapertussis, i.e. BPP0191 and BPP0190 with GenBank™ accession numbers NP_882552 and NP_882551, respectively, and in B. bronchiseptica, i.e. BB0194 and BB0193 with GenBank™ accession numbers NP_886744 and NP_886743, respectively. The mutual sequence identity between the Bordetella proteins is 97% for the LpxL1 proteins and 98% for the LpxL2 proteins. Furthermore, the genetic organization of the lpxL1/lpxL2 operon is conserved among the Bordetella strains. Cloning of lpxL Genes and Complementation of the E. coli lpxL Mutant Phenotype—E. coli lpxL mutants show a growth defect on nutrient broth above 32 °C (14Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (67) Google Scholar). To test whether the identified B. pertussis lpxL homologues can complement this phenotype, we cloned the lpxL1 and lpxL2 genes into the broad host range, low copy number vector pMMB67EH under the control of the tac promoter and used the resulting plasmids to transform E. coli lpxL mutant strain MLK53 (19Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (93) Google Scholar). As controls, both MLK53 and the parental E. coli strain W3110 were transformed with vector pMMB67EH. The strains were first grown to early log phase in synthetic minimal medium at 30 °C, after which the bacteria were transferred to LB and further incubated at 42 °C. The growth defect of the E. coli lpxL mutant was complemented by the plasmid harboring lpxL2 (Fig. 2). The plasmid encoding LpxL1 did not complement the phenotype and its presence, as compared with the empty vector control, seemed to hamper growth even further. Overexpression of lpxL1 and lpxL2 in B. pertussis—The effect on LPS composition of lpxL1 and lpxL2 overexpression was studied in B. pertussis strain B213 after introduction of the pMMB67EH-derived plasmids. No obvious effect of lpxL1 and lpxL2 overexpression was observed upon Tricine-SDS-PAGE analysis of isolated LPS (data not shown). To evaluate possible alterations in LPS composition in more detail, the lipid A moieties of the strains were analyzed by MS in the negative-ion mode. This analysis revealed the presence of two major lipid A species in wild-type LPS (Fig. 3A). The peak at m/z 1557 represents the characteristic penta-acylated bis-phosphate species that is typically found in B. pertussis (22Caroff M. Deprun C. Richards J.C. Karibian D. J. Bacteriol. 1994; 176: 5156-5159Crossref PubMed Google Scholar), whereas the peak at m/z 1477 corresponds to a penta-acylated mono-phosphate species. Besides these two major lipid A species, several minor species were detected. The two peaks at m/z 1307 and 1251 represent deacylated lipid A species of the molecular ion at m/z 1477 that miss the primary 3OH C10 residue at the 3 position and a primary 3OH C14 residue, probably at the 3′ position (30Geurtsen J. Steeghs L. Hamstra H.-J. ten Hove J. de Haan A. Kuipers B. Tommassen J. van

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