INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Feb. 1999, p. 871–878 Vol. 67, No. 2

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Lmb, a Protein with Similarities to the LraI Adhesin Family, MediatesAttachment of Streptococcus agalactiae to Human Laminin

BARBARA SPELLERBERG,1* EVA ROZDZINSKI,2 SIMONE MARTIN,1 JOSEPHINE WEBER-HEYNEMANN,1

NORBERT SCHNITZLER,1 RUDOLF LUTTICKEN,1 AND ANDREAS PODBIELSKI2

Institute of Medical Microbiology, University Hospital Aachen, D-52057 Aachen,1 and Institute ofMicrobiology and Immunology, Hospital of the University, D-89081 Ulm,2 Germany

Received 20 July 1998/Returned for modification 26 August 1998/Accepted 3 November 1998

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular ma-trix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinantsof this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipo-protein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus weredemonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide se-quence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of thecommon serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strainsto laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus wascloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinantLmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmbmediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial coloni-zation of damaged epithelium and translocation of bacteria into the bloodstream.

The expression of cell surface receptors determines adhesiveproperties of streptococci, which include binding to eukaryoticextracellular matrix (ECM) proteins, epithelial cells, and en-dothelial cells, as well as to other bacteria. The LraI (lipopro-tein receptor antigen I) family of surface-associated lipopro-teins is involved in the coaggregation of Streptococcus gordoniiwith Actinomyces naeslundii, the adherence of S. sanguis to thesalivary pellicle, the binding of S. parasanguis to a plateletfibrin matrix (14, 37), and the adherence of S. pneumoniae totype II pneumocytes (3). Previously identified members of thisfamily are PsaA from S. pneumoniae, FimA from S. parasan-guis, SsaB from S. sanguis, EfaA from Enterococcus faecalis,ScbA from S. crista, and ScaA from S. gordonii. Proteins of thisfamily appear to serve a dual role in adhesion and transport;they are located in ABC transporter-type operons and code forlipoproteins. Similarities between the deduced proteins of lraIgenes and MntC, an Mn21 transporter of Synechocystis, havebeen described (1), and recently Mn21 transporter activity wasdemonstrated for PsaA of S. pneumoniae (5) and ScaA ofS. gordonii (17). It has been proposed that the LraI proteinstogether with other proteins constitute a large family ofmetal transporters (5). With regard to pathogenicity, PsaAof S. pneumoniae and FimA of S. parasanguis have been shownto be essential for virulence in animal models (3, 37), andimmunogenic properties were demonstrated for EfaA (19),FimA (37), and PsaA (35), indicating their potential use asvaccine candidates.

S. agalactiae (group B streptococcus [GBS]) is one of themost important neonatal pathogens, causing 1.8 cases of sep-ticemia or meningitis per 1,000 live births (40). Despite ade-quate antimicrobial therapy, mortality rates still range between5 and 30% (38). In addition, recent studies have found anincreasing number of serious infections in adults (7, 8). Several

virulence factors that contribute to the pathogenesis of thedisease have been identified: capsular polysaccharides (39),CAMP factor (9, 28), hemolysin (24), and C proteins (23). Theadherence of S. agalactiae to immobilized fibronectin has beenimplicated in the pathogenesis of disease (34), but geneticdeterminants for the adherence of S. agalactiae to ECM pro-teins have not been identified.

Laminin, a 900-kDa glycoprotein, is a major component ofthe basement membrane. It is composed of three distinct poly-peptide chains (A, B1, and B2) which reversibly assemble toform the macromolecular structure. Functions of laminin in-clude the formation of the basement membrane by interactionwith other basement membrane components and the develop-ment and maintenance of cellular organization. S. agalactiaehas been demonstrated to damage the pulmonary epithelium(24), a process that leads to the exposure of underlying base-ment membrane structures. Thus, the adhesion to basementmembrane components may be critical for the bacterial colo-nization of damaged epithelium and invasion of bacteria intothe bloodstream.

In this paper, we describe the identification of a putativelipoprotein with homology to the streptococcal LraI family inGBS. We show that mutants of the genetic locus are deficientin adherence to immobilized laminin and that the recombinantprotein inhibits adherence of the wild-type strain to humanlaminin.

MATERIALS AND METHODS

Bacterial strains. The Escherichia coli and S. agalactiae strains used in thisstudy are listed in Table 1. E. coli DH5a served as the host for recombinantplasmid pG1host5, E. coli BL21 was used for the expression of recombinantprotein from plasmid pET21a, and E. coli XL1-Blue MRF and XLOLR (Strat-agene, Heidelberg, Germany) were used as hosts for phages Lambda ZAPExpress and ExAssist, respectively.

S. agalactiae isolates were cultured on Columbia agar (Oxoid, Basingstoke,England) supplemented with 3% sheep blood, in Todd-Hewitt broth (THB)(Oxoid) or in THB supplemented with 0.5% yeast (THY) at 37°C. Mutant strainsharboring chromosomally integrated pG1host5 vectors were maintained in me-dium containing 5 mg of erythromycin per liter at a temperature of $37°C.

* Corresponding author. Mailing address: Institute of MedicalMicrobiology, University Hospital Aachen, Pauwelsstr. 30, D-52057Aachen, Germany. Phone (49)-241-8088454. Fax: (49)-241-8888483.E-mail: bspeller@imib.rwth-aachen.de.

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Growth rates of wild-type and mutant strains were determined by measuringoptical density at 600 nm (OD600) in THY or THY supplemented with MnCl2.

General DNA techniques. Standard recombinant DNA techniques were usedfor nucleic acid preparation and analysis. PCR was carried out with Taq poly-merase as specified by the manufacturer (Boehringer, Mannheim, Germany),with 35 cycles of amplification steps of 1 min at 94°C, 1 min at 50 to 56°C, and1 to 3 min at 72°C, depending on product size. For PCR with degenerate primers,PCR conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 45°Cfor 30 s, and 72°C for 1 min, with primer 59-GGG GGG ATC CRT SNN SGAYRA YGG-39 and 59-GGG GGG ATC CAR SCC SAV SCC SNN SC-39 (R 5A or G; S 5 G or C; N 5 A, G, C, or T; Y 5 C or T; V 5 G, A, or C). Genomicstreptococcal DNA was isolated as described previously (22). To confirm thepresence of lmb in different serotypes, Southern blot analysis was performedafter digestion of genomic DNA with EcoRI. Hybridization was performed at65°C overnight. The hybridization probe was generated by PCR of strain R268with primers 59-ACC GTC TGT AAA TGA TGT GG-39 and 59-GAT TGA CGTTGT CTT CTG-39, and the resulting PCR products were labeled by adding Dig(digoxigenin)-dUTP (Boehringer) at a final concentration of 5 mM. Hybridizingfragments were visualized by disodium 3-{4-methoxyspiro[1,2-dioxetane-3,29-(59-chloro)tricyclo[3.3.1.13,7) decan]-4-yl}phenyl phosphate CSPD; Serva, Heidel-berg, Germany) as instructed by the manufacturer. Plasmid DNA was isolatedand purified by using a Qiaprep Spin Miniprep kit (Qiagen, Hilden, Germany)according to the manufacturer’s instructions. Plasmid and PCR products weresequenced on an ABI 373 automated DNA sequencer, using an ABI PRISM dyeterminator cycle sequencing kit (PE Applied Biosystems, Weiterstadt, Germa-ny). GBS strains were transformed according to the protocol of Ricci et al. (30).

Immunofluorescence. For the immunofluorescence test, bacteria were grownovernight in THY. Incubation with anti-Lmb antibody was performed at a dilu-tion of 1:100 in phosphate-buffered saline–2% goat serum for 30 min. Thesecondary antibody fluorescein isothiocyanate-labeled goat anti-rabbit immuno-globulin G) (Sigma Chemical Co., St. Louis, Mo.) was used at a concentration of1:100 in phosphate-buffered saline–2% goat serum. Fluorescence was assessedvisually under a fluorescence microscope and measured in a FACScan flowcytometer (Becton Dickinson, San Jose Calif.) equipped with a standard argonlaser.

Phage techniques. A Lambda ZAP Express library of strain O90R was createdas described by Podbielski et al. (29). Briefly, 200 mg of genomic DNA wasdigested with 0.2 U of Sau3A (Boehringer) for 30 min at 37°C. The resulting

DNA fragments were separated according to size by a salt gradient technique(12). Fractions containing fragments 2 to 9 kb in length were ligated withBamHI-digested l arms and packaged by using a Gigapack II packaging kit(Stratagene). Further processing and plaque lifting were done according to themanufacturer’s instructions. The library was screened by hybridization with PCRproducts at 65°C overnight. The PCR products were labeled by adding Dig-dUTP (Boehringer) at a final concentration of 5 mM to the reaction mixture.Detection of positive plaques by CSPD (Serva) was done as instructed by themanufacturer.

Construction of lmb mutants. Plasmid pG1host5 was used for targeted geneticmutagenesis of lmb. Two mutants of the wild-type strain O90R (Lmb-k1 andLmb-k2) were created by plasmid insertion at nucleotides 495 and 777, respec-tively, of the lmb gene. Internal fragments of the lmb gene were amplified byPCR with primers 59-ACC GTC TGT AAA TGA TGT GG-39 plus 59-GATTGA CGT TGT CTT CTG C-39 and 59-GCC GCC ACTAGT ACC GTC TGTAAA TGA TGT GG-39 plus 59-GAC GAC GAA TTCGAT TGA CGT TGTCTT CTG C-39 (the newly introduced SpeI and EcoRI restriction sites areunderlined). Resulting PCR products and the vector were digested with BamHIand XbaI and with SpeI and EcoRI, respectively, ligated, and transformed into E.coli. Chromosomal integration into strain O90R was performed as previouslydescribed (21). To confirm correct chromosomal insertion of the plasmid,genomic DNA of mutant Lmb-k1 and wild-type strain O90R was digested withthe restriction endonuclease EcoRI or XbaI and probed with a nucleotide probedirected to the duplicated fragment of lmb. PCR with primers annealing tovector sequences and genomic nucleotide sequence upstream or downstream ofthe duplication site followed by DNA sequencing of PCR products was used toconfirm chromosomal insertion for both mutants.

Expression of Lmb in E. coli. The lmb gene was cloned into the pET21aexpression vector (Novagen, Madison, Wis.) in E. coli BL21(DE3) (Novagen) forhigh-level expression and purification over a Ni21 column. To construct thepET21a::lmb vector, nucleotides coding for amino acids 19 to 306 were amplifiedby PCR using primers 59-GCC GCG CAT ATG TGT GAT AAG TCA GCAAAC CCC A-39 and 59-GCC GCG CTC GAG CTT CAA CTG TTG ATA GAGCAC TTC C-39 (the newly introduced restriction sites NdeI and XhoI are un-derlined). The resulting PCR product was purified by using a QiaQuick PCRpurification kit (Qiagen) according to the manufacturer’s instructions. The pu-rified product and pET21a plasmid were digested with NdeI and XhoI, ligated,and introduced into E. coli by using standard molecular biology techniques. For

TABLE 1. Bacterial strains and plasmids used

Strain or plasmid Description Source or referencea

StrainsE. coli

DH5a endA1 hsdR17 supE44 DlacU169(f80lacZDM15) recA1 gyrA96 thi-1 relA1 BoehringerXL1-Blue MRF D (mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1

lac [F9proAB lacIqZDM15 Tn10 (Tetr)]Stratagene

BL21(DE3) E. coli B, F2 dcm ompT hsdS gal l(DE3), T7 polymerase gene under control ofthe lacUV5 promoter

Novagen

S. agalactiaeR268 Serotype III, Hly2 Aachen collectionO90R (ATCC 12386) Serotype Ia, Lancefield grouping strain ATCCATCC 12400 Serotype Ia ATCCATCC 12401 Serotype Ib ATCCATCC 27591 Serotype Ia/c ATCC18 RS 26 Serotype II R. Lancefield collectionCNCTC 13/63 Serotype III CNCTCCNCTC 1/82 Serotype IV CNCTCM 1A-00008 Serotype V P. Ferrieri collection92-085 Serotype VI P. Ferrieri collection

(original NCTC 2/86)87-603 Serotype VII P. Ferrieri collection

(‘7271’ original Perch)JM9-130013 Serotype VIII P. Ferrieri collectionLmb-k1 Strain O90R with pG1host5 integrated into the lmb gene at codon 165 This studyLmb-k2 Strain O90R with pG1host5 integrated into the lmb gene at codon 259 This study

PlasmidspUC18 Apr, ColE1, lacI, f80dlacZ BoehringerpG1host5 Eryr, pBR, Ts AppligenepET21a Apr, T7lac, pBR, His tag NovagenpBS1876 pET21a vector carrying the 918-bp coding region of lmb This studypBS1817 pG1host5 derivative carrying an internal 201-bp fragment of lmb This studypBS1815 pG1host5 derivative carrying an internal 572-bp fragment of lmb This study

a ATCC, American Type Culture Collection; CNCTC, Czechoslovak National Collection of Type Cultures.

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the expression of recombinant protein, E. coli BL21(DE3) (Novagen) harboringthe pET21a::lmb construct was grown to an OD600 of 0.6 in Luria-Bertanimedium, and protein expression was induced by 1 mM isopropyl-b-D-thiogalac-topyranoside (IPTG) for 2 h; cells were pelleted, resuspended in 1 ml of lysisbuffer (50 mM sodium phosphate [pH 8.0], 300 mM NaCl, 20 mM imidazole, 1mg of lysozyme per ml, 1 mM phenylmethylsulfonyl fluoride), placed on ice for30 min, and subjected to sonication. Recombinant Lmb was purified from lysedE. coli cells by passage over a commercial nickel affinity matrix (Ni-NTA [ni-trilotriacetic acid] Spin kit; Qiagen) and eluted under native conditions accordingto the manufacturer’s instructions. The eluate was subjected to 8 to 25% gradientsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) usingthe Phast system (Pharmacia LKB, Uppsala, Sweden) and visualized by silverstaining.

Subcellular localization. Polyclonal antibodies for Lmb were obtained fromEurogentec (Brussels, Belgium). Antibodies were raised in New Zealand Whiterabbits by intradermic injection of 100 mg of recombinant Lmb at days 0, 14, 28,and 56. Subcellular fractions were prepared from GBS strain O90R. Bacteriawere grown to late logarithmic phase in THB supplemented with 3% sheepblood. Cells were disrupted at a pressure of 16,000 to 20,000 PSI lb/in2 in ahigh-pressure homogenizer (Avestin Inc., Ottawa, Ontario, Canada); cytoplas-mic and membrane fractions were separated by centrifugation at 100,000 3 g for60 min. Cytoplasmic proteins were precipitated by trichloroacetic acid, whereasthe pellets containing the membranes were used directly. Samples of both frac-tions containing 6 mg of protein each were solubilized in SDS-gel electrophoresisbuffer, separated by denaturing SDS-PAGE on a 8 to 25% gradient gel, andtransferred to an Immobilon P polyvinylidene difluoride membrane (Millipore,Eschborn, Germany) with the Phast system (Pharmacia LKB) according to themanufacturer’s instructions. The blots were probed with a polyclonal anti-Lmbantibody at a dilution of 1:1,000. Controls were probed with preimmune rabbitserum at a dilution of 1:1,000. Primary antibodies were detected by an alkalinephosphatase-labeled anti-rabbit immunoglobulin G secondary antibody (Pierce,St. Augustin, Germany) at a dilution of 1:5,000. Bound secondary antibodieswere visualized by chemiluminescent CSPD (Serva) according to the manufac-turer’s instructions.

RNA preparation and analysis. Total RNA was prepared from GBS strainsR268 grown to an OD of 0.8 in THB. Cells were lysed mechanically by glassbeads in a cell disrupter (Dianova, Hamburg, Germany) in the presence of 1 mlof Trizol (Gibco BRL, Eggenstein, Germany). Purification of the RNA was doneaccording to the manufacturer’s instructions. Reverse transcription RT was car-

ried out with 1 mg of RNA as template, 2 pmol of primer 59-GCAGCAGCAGCAGGACAGCACTGATTTGATCC-39, 0.1 M dithiothreitol, 10 mM deoxy-nucleoside triphosphate mix, and 200 U of Superscript II reverse transcriptase(Gibco BRL) in 50 mM Tris-HCl (pH 8.3)–75 mM KCl–3 mM MgCl2 at 42°C for50 min in a 20-ml reaction volume; 5 ml of the reaction mixture was used as thetemplate for a subsequent PCR with primers 59-ACCGTCTGTAAATGATGTGG-39 and 59-CAGCACTGATTTGATCC-39.

Adherence assay. To study adherence of S. agalactiae wild-type strain O90Rand lmb mutant strains Lmb-k1 and Lmb-k2 to immobilized laminin, 60-wellTerasaki plates were coated with human placental laminin (100 mg/ml; GibcoBRL) reconstituted in Dulbecco’s phosphate-buffered saline (DPBS). Plateswere incubated with laminin for 18 h at room temperature. Streptococci weregrown in THB, harvested in mid-logarithmic phase, and washed twice inDPBS. Bacteria were labeled with fluorescein isothiocyanate and resuspended inDPBS. To obtain single cells, bacterial suspensions were sonicated. Laminin-coated wells were washed with DPBS, and then 10 ml of DPBS containing 5 3106 bacteria was added to each well. For the Mn21 supplementation studies,MnCl2 was added to a concentration of 10 mM to DPBS. To investigate the effectof recombinant Lmb on the adherence of the wild-type strain O90R, Terasakiwells were preincubated for 20 min at 37°C with 1 mg of recombinant Lmb beforethe bacterial suspension was added. After incubation for 60 min at 37°C, non-adherent bacteria were removed by being washed five times with DPBS. Adher-ent bacteria were quantified in a fluorescence counter (Cytofluor II; PerseptiveBiosystems Inc.).

Nucleotide sequence accession number. The nucleotide sequence of the cod-ing regions for the S. agalactiae lmb gene has been submitted to the EMBL/Genbank/DDBJ nucleotide sequence data libraries and assigned accession no.AF062533.

RESULTS

Identification of Lmb. Fragments of chromosomal DNAfrom GBS strain R268 were amplified by PCR using degener-ate primers directed toward the conserved glycine-rich G1 andG2 blocks of bacterial sensor proteins, which resemble nucle-otide binding domains (27). This method has previously beenused to amplify the nucleotide sequence of cell surface-asso-

FIG. 1. Amino acid sequence alignment of LraI proteins (S. pneumoniae PsaA, S. parasanguis FimA, S. sanguis SsaB, S. gordonii ScaA, S. crista ScbA, and E. faecalisEfaA) and S. agalactiae Lmb. Alignment and determination of consensus sequence were performed with the MultAlin program (http://www.toulouse.inra.fr). Aminoacid residues of Lmb matching the consensus sequence are shown in boxes. Highly conserved residues (consensus level of 90%) are represented as capital letters inthe consensus sequence; small letters denote a consensus level of $50%. !, I or V; $, L or M; %, F or Y; #, N, D, Q, E, or B. Parameters: gap weight, 12; gap lengthweight, 2.

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ciated transport proteins in gram-positive organisms (2). Theresulting PCR products were purified and subcloned into plas-mid pUC18. Nucleotide sequences of the inserts were deter-mined by automated DNA sequencing. Comparison of the de-duced amino acid sequence with the GenBank database entriesrevealed that 3 of 42 clones harbored overlapping fragmentsof a gene with significant homology to the streptococcal LraIprotein family.

Nucleotide and protein sequence analysis. Nucleotide se-quence upstream and downstream of the initial chromosomalfragments was obtained by screening of a l phage library.Primers to generate PCR products of strain R268 weredesigned based on the nucleotide sequence of positive plaques.DNA sequencing of the resulting PCR products revealed anopen reading frame of 921 nucleotides with a typical ribosomebinding site 5 nucleotides upstream of the ATG start codon.Based on sequence similarity, a putative prokaryotic 235 and210 promoter region was identified approximately 70 nucleo-tides upstream of the start codon. The deduced protein con-sists of 306 amino acid residues with a predicted molecularmass of 34.1 kDa. A second open reading frame of 2.4 kb starts12 nucleotides downstream of the lmb stop codon. The puta-tive start codon, GTG, is preceded by a typical ribosome bind-ing site (GAAGGA). Putative promoter sequences could notbe identified in the short intergenic region between lmb andthe downstream open reading frame or within the 39-terminalregion of the lmb gene, suggesting that the two genes maycomprise an operon. All of the known LraI proteins are li-poproteins with the typical signal peptidase II recognition se-quence LxxC for the signal peptidase II at amino acid residue16 or 17. The GBS homologue has a similar but slightly dif-ferent sequence at this position (IAGC), with a leucine-to-isoleucine alteration. LplA of Bacillus subtilis, which has beenshown to be a lipoprotein by radiolabeling with palmitate, alsocontains this atypical recognition sequence (32).

Comparison of the deduced amino acid sequence with se-quences of previously identified members of the LraI adhesinfamily revealed 47% homology and 27% identity with PsaA ofS. pneumoniae. Similarities to the other LraI proteins werebetween 36 and 46% (Fig. 1). In addition, there was 30%identity with Adc, a novel Zn transporter of S. pneumoniaewhich presumably binds Zn through a histidine-rich region ofthe protein. Lmb, however, does not possess a similar domain.Jenkinson (14) proposed four common structural domains ofthe LraI proteins: a 20-residue hydrophobic leader sequencethat is cleaved off by signal peptidase II; two transmembranedomains, B1 and B2; and the a region, which is assumed to beexposed to the cell surface and comprises the solute bindingregion of the protein. These domains appear to be conservedin the lmb gene product (Fig. 2). Interestingly, amino acidresidues 152 to 197 of Lmb, corresponding to the a domain ofthe LraI family, exhibit 50% homology to the human lamininB2 chain (Fig. 2).

Presence and expression of the lmb gene in various GBSserotypes. To determine the distribution of lmb in variousS. agalactiae strains, a fragment of the gene was amplified byPCR and used as a hybridization probe for a Southern blot ofGBS serotypes Ia, Ib, Ic, and II to VIII. Hybridization of theprobe with chromosomal DNA could be detected as a singleband in all of the serotypes tested (Fig. 3); immunofluores-cence tests performed on the different serotypes confirmed theexpression of the protein in these strains.

Transcription analysis of the lmb locus. Several of the ho-mologous streptococcal lraI genes were reported to be poly-cistronicly transcribed (3, 10, 13, 18). To analyze transcriptionof the lmb locus in GBS, RT-PCR was performed with RNAisolated from strain R268. For the RT reaction, we used areverse primer which anneals to nucleotides 191 to 174 of thesecond open reading frame. The subsequent PCR amplified a930-bp product consisting of the last 716 nucleotides of lmb,the intergenic region, and the first 191 nucleotides of the sec-ond open reading frame (Fig. 4), which supports the hypothesisthat lmb and the second open reading frame are transcribedtogether. To confirm that the specific PCR product originatedfrom mRNA, controls were performed on a portion of theRNA preparation that had not been subjected to an RT reac-tion.

Subcellular localization of Lmb. Based on the homology ofthe deduced protein with other members of the LraI adhesinfamily and the presence of a putative signal peptidase II rec-ognition site, we hypothesized that Lmb is localized at the

FIG. 2. (A) Comparison of Lmb with the LraI domains. The Lmb protein(A) is designated by the open box showing a putative cleavage site for signalpeptidase II. At amino acids (aa) 165 and 229, the plasmid insertion sites of thepG1host5 vector in mutants Lmb-k1 and Lmb-k2 are indicated. A region cor-responding to the a region of LraI proteins with similarity to the B2 chain ofhuman laminin is represented by a filled rectangle. (B) Structural features of theLraI family as proposed by Jenkinson (14). Numbers refer to amino acid resi-dues, demarcating four regions: leader peptide, cleaved off by signal peptidase II;B1; B2; and the a region, which is presumed to be the solute binding domain. (C)Amino acid sequence alignment of the a region of Lmb with the laminin B2chain. The alignment was performed with the BLASTp program at the NationalCenter for Biotechnology Information web site.

FIG. 3. (A) Analysis of the lmb gene in various GBS serotypes by Southernhybridization. Genomic DNA was digested with EcoRI and transferred to anylon membrane. Hybridization was performed with a Dig-dUTP-labeled frag-ment of lmb generated by PCR. Lanes: M, molecular size markers (in nucleo-tides); 1, GBS strain O90R; 2 to 11, specific serotypes as indicated above thelanes. (B) Southern analysis of GBS strain O90R and the isogenic mutantLmb-k1. Genomic DNA of the parent (lanes 1 and 2) and (lanes 3 and 4) mutantstrains was digested with EcoRI (lanes 1 and 3) or XbaI (lanes 2 and 4).Hybridization was performed with a probe directed to the internal fragment oflmb that was used for insertion duplication mutagenesis.

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surface of the bacterial cell. The subcellular localization wasdetermined by Western blot analysis with a Lmb-specific anti-body after cytoplasmic and membrane fractions of the cellswere separated by ultracentrifugation. A single band in themembrane fraction corresponded to the predicted molecularmass of 34 kDa and the size of the recombinant Lmb protein(Fig. 5). Two smaller bands, probably representing degrada-tion products, are present in the lane with the recombinantprotein. Preimmune rabbit serum did not react with controlblots. The results demonstrate that Lmb is associated with thebacterial membrane fraction. Surface exposure of the proteinwas investigated with an immunonofluorescence test. Anti-Lmb antibodies were used to detect Lmb on the surface ofintact bacterial cells, and a fluorescence-labeled secondary an-tibody was used to visualize the binding of anti-Lmb to thebacteria. Results demonstrate that the protein is located on thesurface and that the most intense fluorescence staining is seenat the margins of the cells (Fig. 6).

Construction of lmb mutants by insertion duplication mu-tagenesis. The plasmid pG1host5 (4) was used for targetedgenetic mutagenesis of lmb. Two mutants of the wild-typestrain O90R (Lmb-k1 and Lmb-k2) were created by insertionduplication mutagenesis at nucleotides 495 and 777, respec-tively, of the lmb gene (Fig. 2). Correct chromosomal insertionof the plasmid was confirmed by either Southern blot hybrid-ization (Fig. 3) or PCR and subsequent DNA sequencing ofboth mutants.

Growth properties of S. agalactiae upon Mn21 substitution.It was recently reported that the reduced growth rates of psaAmutants which could be improved by the addition of micromo-lar concentrations of Mn21 to the culture medium (5). There-fore, growth of the lmb mutants in regular THY and THYmedium supplemented with Mn21 was determined by ODmeasurements. Growth rates and final growth densities of thewild-type and mutant strains showed no significant differencesif cultured in THY or in THY supplemented with Mn21 tofinal concentrations of 3 and 10 mM (data not shown). Theseresults indicate that the Mn21 growth requirements are satis-fied in THY, which contains approximately 1 mM Mn21, andthat adhesion deficits of lmb mutants are not attributable toimpaired growth rates.

Adherence to ECM proteins. Since several LraI proteinswere reported to be adhesins and Lmb exhibits homologies tothe human laminin B2 chain, we tested the adherence of GBSwild-type and mutant strains to immobilized human placentallaminin. Adherence of the two independently derived lmb mu-tants (Lmb-k1 and Lmb-k2) was significantly less than that ofthe wild-type strain O90R, in both cases reaching only 25% ofthe wild-type level (Fig. 7). Reduced adherence of lmb mutantswas found with a wide range of bacterial concentrations andwas consistent across incubation times with human lamininranging from 30 to 300 min. To investigate the possibility thatbinding to the immobilized laminin is mediated by a contam-inant of the laminin preparation, we tested the binding ofwild-type and mutant strains to collagen IV, which is present intrace amounts in the laminin preparation. Neither the wild-type nor the mutant strain exhibited significant binding tocollagen IV. Binding of the wild-type strain to collagen IV wasless than 2% of the binding observed for laminin (data notshown). A screen for any major contaminants in the lamininpreparation performed by protein gel electrophoresis and sub-sequent silver staining did not reveal the presence of any un-expected proteins bands (data not shown).

Influence of recombinant Lmb protein on the adherence ofthe wild-type strain. To test the hypothesis that Lmb proteinitself interacts with human laminin, a recombinant protein wasgenerated. The recombinant protein could be visualized as aband of approximately 34 kDa upon SDS-PAGE, which isconsistent with the predicted molecular mass of Lmb (Fig. 8).To evaluate the influence of Lmb on adherence of the wild-type strain, the immobilized laminin was incubated with re-combinant protein prior to adherence assays. Preincubationwith recombinant Lmb reduced the adherence of the wild-typestrain significantly, to 60% of the initial values (Fig. 7). Inter-estingly, adherence of the isogenic lmb mutants was slightlyincreased under the same conditions, possibly because the re-combinant protein functions as a bridging molecule betweenthe mutants and laminin.

FIG. 4. Transcription analysis of the lmb locus by RT-PCR. RNA was ex-tracted from S. agalactiae R268 and subjected to RT-PCR. Lanes: 1, DNA sizemarker; 2, PCR with chromosomal DNA as the template; 3, PCR with 1 mg ofRNA as the template; 4, control for DNA contamination in which the RNApreparation was subjected to PCR without prior RT-PCR.

FIG. 5. Subcellular localization of Lmb. Bacterial cells were disrupted by ahigh-pressure cell homogenizer, separated into membrane and cytoplasmic frac-tions by centrifugation at 100,000 3 g, and subjected to denaturing gel electro-phoresis. Western immunoblotting was performed as described in Materials andMethods with a polyclonal anti-Lmb antibody (A) or preimmune serum (B) at adilution of 1:1,000. Lane 1, cytoplasmic fraction; 2, membrane fraction; 3, re-combinant Lmb (3 ng).

FIG. 6. Analysis of the surface exposure of Lmb by immunofluorescencestaining. S. agalactiae cells were labeled with a polyclonal anti-Lmb antibody andviewed by fluorescence microscopy.

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Influence of Mn21 supplementation on adherence. To in-vestigate if supplementation with Mn21 affected the adherenceof bacteria to immobilized laminin, the wild-type strain andmutant strain Lmb-k1 were grown in THB or THB supple-mented to a concentration of 10 mM Mn21 and then subjectedto the adherence assay in the presence of 10 mM Mn21. Underthese conditions, adherence of the mutant strain remainedsignificantly less than that of the wild-type strain (Fig. 7); thelevel for the mutant reached 34% of the wild-type level, dem-onstrating that the effect on adherence to immobilized humanlaminin cannot be circumvented by growing cells in the pres-ence of excess Mn21 or adding Mn21 to the adherence assay.

DISCUSSION

Pathogenic bacteria frequently express surface proteins thatadhere to components of the mammalian ECM. The interac-tion of microorganisms with ECM proteins can promote bac-terial colonization of damaged tissues. Despite considerable

FIG. 7. Adherence of S. agalactiae wild-type (O90R) and lmb mutant (Lmb-k1 and Lmb-k2) strains to immobilized laminin. Adherence was tested in Terasaki wellscoated with 100 mg of ECM protein per ml. Assays were carried out as described in Materials and Methods. Bars represent the mean 6 standard deviation for six wells.Results are representative for at least three independent experiments performed for each strain. (A) Adherence to laminin with increasing incubation time; (B)adherence to laminin for different bacterial inocula; (C) adherence to laminin after preincubation of immobilized laminin with recombinant Lmb protein; (D) adherenceto laminin upon Mn21 substitution.

FIG. 8. SDS-PAGE analysis of recombinant Lmb. Bacterial lysates and pu-rified recombinant Lmb were separated on an 8 to 25% SDS-polyacrylamide gelalong with molecular mass markers and silver stained. Lanes 1, molecular massmarker; 2, crude bacterial lysate of E. coli BL21(DE3)(pET21a::lmb), unin-duced; 3, crude bacterial lysate of E. coli BL21(DE3)(pET21a::lmb) after induc-tion of protein expression with IPTG; 4, recombinant Lmb after purification overa Ni21-NTA affinity matrix.

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understanding of the molecular mechanisms of this process inother streptococcal species, genetic determinants for the ad-herence of S. agalactiae to ECM proteins have not been iden-tified. In this investigation, we characterized the gene encodinga protein of S. agalactiae with similarities to the LraI adhesinfamily that mediates the attachment of GBS to laminin andwas subsequently designated lmb. Comparison of the deducedamino acid sequence with LraI proteins showed closest homol-ogies to PsaA of S. pneumoniae. However, compared to theconservation between proteins of this family present in otherstreptococci, the gene of GBS is distinct. Within the group oforal streptococci and the genetically related S. pneumoniae,similarities range between 80 and 93%. The results could re-flect the more distant relationship of b-hemolytic streptococciwith other streptococcal groups or indicate that the GBS pro-tein has diverged from other LraI proteins.

We found that Lmb is important for the laminin bindingproperties of S. agalactiae. Isogenic mutants of the lmb locusdemonstrated substantially diminished adherence to immobi-lized laminin, and the recombinant protein inhibited attach-ment of the wild-type strain. Since the mutants were generatedby insertion duplication mutagenesis, the inserted plasmid canlead to polar effects on downstream genes; we cannot rule outthat these effects may contribute to some extent to the ob-served phenomenon. However, the reduction of wild-type ad-herence by the recombinant protein is a strong indication thatthe lmb gene product directly mediates the interaction betweenGBS and laminin.

FimA from S. parasanguis and ScaB from S. gordonii havebeen shown to be important for the binding of these microor-ganisms to saliva-coated hydroxylapatite (10, 13). Glycopro-teins of the saliva are assumed to be the ligands for theseadhesins. Analogous to these findings, our results indicate thatLmb binds to laminin, a high-molecular-weight glycoprotein ofthe basement membrane. The similarity of the putative a re-gion of Lmb to the human laminin B2 chain could account foran interaction with other polypeptide chains of the lamininmolecule. Laminin is a macromolecule that self-assembles invitro to form large unordered aggregates and can bind a varietyof other basement membrane compounds, such as nidogen,collagen IV, and perlecan (6). Interestingly psaA mutants ofS. pneumoniae exhibit reduced binding to A549 cells (3), a typeII pneumocyte cell line that secrects laminin (16).

The adherence of bacteria to laminin may be a crucial stepin the development of invasive GBS infection. Translocation ofS. agalactiae into the bloodstream as well as entry of the bac-teria into the cerebrospinal fluid, which occurs in the case ofmeningitis, requires the passage of bacteria through the base-ment membranes. The interaction of bacterial surface proteinswith laminin could be important in this context. In the case ofHaemophilus influenzae, adhesion and penetration of the base-ment membranes, which are open to circulation in the fenes-trated endothelium of the choroid plexus, is discussed as theroute of entry into the cerebrospinal fluid (36).

The results of the RT-PCR experiments show that lmb ispart of an operon and that it is transcribed together with asecond open reading frame, similar to the operon structure ofother members of this family (10, 13, 31). Several differentobservations indicate that besides playing a role in adhesionand virulence, proteins of the LraI family function as trans-porters. Transport systems of gram-positive bacteria usually con-tain a solute binding component that is lipid modified andassociated with the outer region of the cytoplasmic membrane(33). The fimA locus of S. parasanguis encodes an ATP bindingmembrane transport system (11), and manganese transporter

activity has been demonstrated for PsaA of S. pneumoniae (5)and ScaA of S. gordonii (17).

It has been suggested that all members of the LraI family aremanganese transporters and that the inhibition of S. sanguisand S. parasanguis adhesion to saliva-coated hydroxylapatite bypurified ScaA or FimA is not due to direct binding of theseproteins but rather reflects a requirement for Mn21 (5). Toinvestigate if the effects of an Lmb mutation are influenced bythe availability of Mn21, culture of bacterial cells and adher-ence assays were performed under supplementation withMn21. Mutants grown in THB containing approximately 1 mMMn21 and THB supplemented to an Mn21 concentration of 10mM both exhibited significantly reduced adherence to immo-bilized laminin, demonstrating that the reduced binding prop-erties cannot be explained by Mn21 deficiency alone. Ourresults indicate a function of Lmb in the adhesion to lamininthat is distinct from the putative role as a Mn21 transporter.

Immunogenic properties have been demonstrated for threemembers of the LraI adhesin family, PsaA, EfaA, and FimA (3,19, 37). A recent publication demonstrates induction of pro-tective antibodies against S. parasanguis endocarditis in ananimal model after vaccination with recombinant FimA (37).Current vaccine approaches for GBS favor the use of proteinslinked to polysaccharide structures of the capsule (15, 20, 25,26). None of the presently identified surface proteins of GBSare found in every known serotype; given the presence of Lmbin all of these serotypes, the protein may be a good candidatefor GBS vaccine.

ACKNOWLEDGMENTS

We thank P. Ferrieri for generously providing S. agalactiae strainsand W. Dott for the determination of Mn21 in THY and THB. We aregrateful to B. Leonard and C. Brandt for helpful discussions andcritical review of the manuscript.

This work was supported by grants from the Deutsche Forschungs-gemeinschaft (Sp 511/2-1 and Po 391/6-1) to B.S. and A.P.

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Editor: E. I. Tuomanen

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