Lmb, a Protein with Similarities to the LraI Adhesin Family, Mediates Attachment of <em>Streptococcus agalactiae</em> to Human Laminin

B Spellerberg

E Rozdzinski

S Martin

J Weber-Heynemann

N Schnitzler

R Lütticken

A Podbielski

Institute of Medical Microbiology, University Hospital Aachen, D-52057 Aachen,^{and Institute of Microbiology and Immunology, Hospital of the University, D-89081 Ulm,}^Germany

^{Corresponding author. Mailing address: Institute of Medical Microbiology, University Hospital Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany. Phone (49)-241-8088454. Fax: (49)-241-8888483. E-mail:}ed.nehcaa-htwr.bimi@rellepsb.

Received 1998 Jul 20; Revisions requested 1998 Aug 26; Accepted 1998 Nov 3.

Abstract

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

Abstract

The expression of cell surface receptors determines adhesive properties of streptococci, which include binding to eukaryotic extracellular matrix (ECM) proteins, epithelial cells, and endothelial cells, as well as to other bacteria. The LraI (lipoprotein receptor antigen I) family of surface-associated lipoproteins is involved in the coaggregation of Streptococcus gordonii with Actinomyces naeslundii, the adherence of S. sanguis to the salivary pellicle, the binding of S. parasanguis to a platelet fibrin matrix (14, 37), and the adherence of S. pneumoniae to type II pneumocytes (3). Previously identified members of this family are PsaA from S. pneumoniae, FimA from S. parasanguis, SsaB from S. sanguis, EfaA from Enterococcus faecalis, ScbA from S. crista, and ScaA from S. gordonii. Proteins of this family appear to serve a dual role in adhesion and transport; they are located in ABC transporter-type operons and code for lipoproteins. Similarities between the deduced proteins of lraI genes and MntC, an Mn^{transporter of}Synechocystis, have been described (1), and recently Mn^{transporter activity was demonstrated for PsaA of}S. pneumoniae (5) and ScaA of S. gordonii (17). It has been proposed that the LraI proteins together with other proteins constitute a large family of metal transporters (5). With regard to pathogenicity, PsaA of S. pneumoniae and FimA of S. parasanguis have been shown to be essential for virulence in animal models (3, 37), and immunogenic properties were demonstrated for EfaA (19), FimA (37), and PsaA (35), indicating their potential use as vaccine candidates.

S. agalactiae (group B streptococcus [GBS]) is one of the most important neonatal pathogens, causing 1.8 cases of septicemia or meningitis per 1,000 live births (40). Despite adequate antimicrobial therapy, mortality rates still range between 5 and 30% (38). In addition, recent studies have found an increasing number of serious infections in adults (7, 8). Several virulence factors that contribute to the pathogenesis of the disease have been identified: capsular polysaccharides (39), CAMP factor (9, 28), hemolysin (24), and C proteins (23). The adherence of S. agalactiae to immobilized fibronectin has been implicated in the pathogenesis of disease (34), but genetic determinants for the adherence of S. agalactiae to ECM proteins have not been identified.

Laminin, a 900-kDa glycoprotein, is a major component of the basement membrane. It is composed of three distinct polypeptide chains (A, B1, and B2) which reversibly assemble to form the macromolecular structure. Functions of laminin include the formation of the basement membrane by interaction with other basement membrane components and the development and maintenance of cellular organization. S. agalactiae has been demonstrated to damage the pulmonary epithelium (24), a process that leads to the exposure of underlying basement membrane structures. Thus, the adhesion to basement membrane components may be critical for the bacterial colonization of damaged epithelium and invasion of bacteria into the bloodstream.

In this paper, we describe the identification of a putative lipoprotein with homology to the streptococcal LraI family in GBS. We show that mutants of the genetic locus are deficient in adherence to immobilized laminin and that the recombinant protein inhibits adherence of the wild-type strain to human laminin.

ACKNOWLEDGMENTS

We thank P. Ferrieri for generously providing S. agalactiae strains and W. Dott for the determination of Mn^{in THY and THB. We are grateful to B. Leonard and C. Brandt for helpful discussions and critical review of the manuscript.}

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

ACKNOWLEDGMENTS

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